Monospecific and multispecific antibodies and method of use

ABSTRACT

This invention relates to monospecific and multispecific antibodies that may be utilized for the diagnosis and treatment of various diseases. In addition, these antibodies may be modified by protease cleavage. Protease control or regulation may be provided by a protease site located in, for example, a linker. These protease-regulated antibodies may also be utilized for the diagnosis and treatment of various diseases.

This application is a national phase application of PCT/EP2008/006750,which was filed on Aug. 16, 2008 and published in English on Feb. 19,2009. PCT/EP2008/006750 claims the benefit of U.S. ProvisionalApplication Ser. No. 60/955,912, filed Aug. 15, 2007, and U.S.Provisional Application Ser. No. 60/955,913, filed Aug. 15, 2007, thecontents of which are incorporated herein by reference in theirentireties.

This application incorporates by reference the contents of a 277 kb textfile created on Feb. 2, 2010 and named“WO2009021754_sequencelisting.txt,” which is the sequence listing forthis application.

FIELD OF THE INVENTION

This invention relates to monospecific and multispecific antibodies thatmay be utilized for the diagnosis and treatment of various diseases. Inaddition, these antibodies may be modified by protease cleavage.Protease control or regulation may be provided by a protease sitelocated in, for example, a linker. These protease-regulated antibodiesmay also be utilized for the diagnosis and treatment of variousdiseases.

BACKGROUND OF THE INVENTION

An antibody may be directed against one or more different antigens orone or more different epitopes on the same antigen. For example, abispecific antibody is directed against two different antigens or twodifferent epitopes on the same antigen. As bispecific antibodies cansimultaneously bind to two distinct targets, these antibodies have greatpotential for antibody-based diagnosis and for the treatment of variousdiseases and disorders such as cancer, infectious diseases, autoimmunediseases, and blood diseases. For example, bispecific antibodies canselectively stimulate and expand T lymphocytes (Wong, et al., J.Immunol. 139:1369-1374, 1987; Wong, et al., Clin. Immunol. Immunopathol.58:236-250, 1991), direct immune cells or toxic agents to kill tumorcells (Lum, et al., Exp. Hematol. 34:1-6, 2006; Wolf, et al., DrugDiscov. Today 10:1237-1244, 2005; Cao, et al., Adv. Drug Deliv. Rev.55:171-197, 2003; Talac, et al., J. Biol. Regul. Homeost. Agents14:175-181, 2000), and simultaneously block two receptors (Lu, et al.,J. Biol. Chem. 279:2856-2565, 2004). In addition, a bispecific antibodymay be used as a substitute for Factor VIII to enhance enzymaticreaction (US Patent Application No. 2007/0041978) or to direct stemcells to the site of injury in patients with myocardial infarction (Lum,et al., Blood Cells Mol. Dis. 32:82-87, 2004).

Bispecific antibodies targeting tumor-associated antigens and toxicagents may be used in cancer therapy. For example, using thistechnology, one arm of the bispecific antibody may be directed to atumor-associated antigen such as Her2, EGF receptor, CD20, CD22, CD30,CD33, CD52, and CA-125, and the other arm of the bispecific antibody maytarget a toxin, drug, or cytokine. That is, bispecific antibodies mayselectively direct toxic agents to tumor cells enhancing the efficacy oftherapeutic antibodies and decreasing systemic toxicity. Examples oftoxin/drug include calicheamicin, doxorubicin, epirubicin, methotrexate,ricin A, saporin, gelonin, and vinca alkaloids, and cytokine examplesinclude tumor necrosis factor alpha (TNF-alpha) and IL-2.

Specific cleavage by proteases of defined sites in biologicallyimportant effector proteins is a well known method for the naturalcontrol of cellular and extracellular physiological processes. Examplesinclude protease activation and inhibition of the coagulation cascade(Butenas, et al., Biochemistry 67:3-12, 2002; Esmon, Chest, 124:26S-32S,2003), protease activation of protease-activatable receptors (Coughlin,Arterioscler. Thromb. Vasc. Biol. 18:514-518, 1998), protease release ofmembrane associated cytokines (Amour, et al., FEBS Lett. 435:39-44,1998), protease processing of prohormones in secretory vesicles (Moore,et al., Arch. Physiol. Biochem. 110:16-25, 2002), and proteaseprocessing of proproteins during secretion (Scamuffa, et al., FASEB J.20:1954-1963, 2006). Proteases are often expressed or located in atissue-specific or tumor-specific manner and examples include themembrane serine protease corin in heart tissue (Yan, et al., Proc. Natl.Acad. Sci. USA 97:8525-8529, 2000), the kallikrein serine proteaseprostate-specific antigen (PSA) in prostate tissue, prostate cancer, andseminal fluid (Veveris-Lowe, et al., Semin. Thromb. Hemost. 33:87-99,2007), the membrane serine protease hepsin in liver tissue and tumors(Xuan, et al., Cancer Res. 66:3611-3619, 2006), coagulation proteasefactor X expressed in the liver and secreted into blood (Miao, et al.,J. Biol. Chem. 267:7395-7401, 1992), and digestive proteases expressedin the pancreas and released to the duodenum (Belorgey, et al., Biochem.J. 313:555-560, 1996). Specific cleavage of amino acid sequences byhuman proteases include thrombin (Chang, Eur. J. Biochem. 151:217-224,1985), factor Xa (Nagai, et al., Methods Enzymol. 153:461-481, 1987),furin (Brennan, et al., FEBS Lett. 347:80-84, 1994), subtilisin-likeprohormone convertases (Lipkind, et al., J. Biol. Chem. 270:13277-13284,1995), and the matrix metalloproteinases (Minod, et al., J. Biol. Chem.281:38302-38313, 2006). Genes encoding specific proteases may beup-regulated in tumor tissue and Table 2 indicates proteases that areassociated with cancer tissue.

Protease cleavage is widely used in in vitro studies to specificallyremove protein or peptide tags from recombinant proteins or to processhybrid recombinant proteins. For example, human rhinovirus 3C protease,thrombin, or factor Xa have been used to remove glutathioneS-transferase (GST) tags (Dian, et al., Life Sciences News—AmershamBiosciences 10:1-5, 2002) and factor Xa has been use to process hybridproteins (Nagai, et al., 1987). Proteases are often targets for drugs asa means to regulate biological processes; and examples include factor Xa(Phillips, et al., J. Med. Chem. 41:3557-3562, 1998), thrombin (Riester,et al., Proc. Natl. Acad. Sci. USA 102:8597-8602, 2005), urokinase(Killeen, et al., Br. J. Cancer 96:262-268, 2007), and factor VIIa(Kohrt, et al., Bioorg. Med. Chem. Lett. 15:4752-4756, 2005). Finally,proteins developed as biological drugs may be modified to preventcleavage by proteases and to improve their stability in vitro or in vivo(Light, et al., Eur. J. Biochem. 262:522-533, 1999; Saenko, et al.,Haemophilia 12:42-51, 2006).

Specific protease cleavage sites have been incorporated into linkersthat link a toxin molecule to a targeting antibody in order to allowprotease specific release of the toxin by intracellular proteases(Trail, et al., Cancer Immunol. Immunother. 52:328-337, 2003).Furthermore, targeting antibodies have been created in many formats. Forexample, bispecific antibodies have been developed to allow binding totwo different antigens or two different epitopes of an antigen by asingle antibody molecule (Segal, et al., Curr. Opin. Immunol.11:558-562, 1999; Tomlinson, et al., Methods Enzymol. 326:461-479, 2000;Wu, et al., Nat Biotechnol. 25:1290-1297, 2007). Other bispecificmolecules have been generated with the ability to block two receptors(Lu, et al., J. Biol. Chem. 279:2856-2865, 2004) and to recruit immunecells to attack cancer cells and tumor tissue (Loffler, et al., Leukemia17:900-909, 2003; Lum, et al., Exp. Hematol. 34:1-6, 2006).

The present invention relates to a novel antibody format, for example,monospecific and multispecific antibodies. The antibodies of the presentinvention may be constructed by tandem linking of two different heavychain (H) variable region domains (V_(H)) and two different light chain(L) variable region domains (V_(L)). The heavy chain and light chain mayform a Fab-like or IgG-like molecule through the disulfide bond betweenconstant (C) regions. Multispecific antibodies may be generated bylinking more than two antibody variable domains.

The antibodies of the present invention may be modified by proteasecleavage. These protease-regulated antibodies may be, for example,monospecific antibodies, bispecific antibodies, or antibodies withsequential binding-activity upon protease digestion in either, forexample, Fab-like or IgG-like format. Protease control or regulation maybe provided by a protease site located in, for example, a linker. Theseprotease-regulated antibodies may be utilized for the diagnosis andtreatment of various diseases, and provide an additional level ofcontrol for biological drugs for therapeutic or diagnostic applications.

DESCRIPTION OF THE FIGURES

FIG. 1. Schematic drawing of a monospecific protease-regulated antibodywith a linker which contains a protease site between variable domain andFc domain (“Type 1”).

FIG. 2. Schematic drawing of a bispecific protease-regulated antibodywith a linker which contains a protease cleavage sequence that allowsremoval of one antigen-binding site (“Type 2”).

FIG. 3. Schematic drawing of another bispecific protease-regulatedantibody with a linker which contains a protease cleavage sequence thatallows removal of one antigen-binding site (“Type 2”).

FIG. 4. Schematic drawing of the application of a bispecificprotease-regulated antibody that simultaneously binds two differentantigens.

FIG. 5. Schematic drawing of a protease-regulated antibody that cannotbind to two different antigens simultaneously (“Type 3”).

FIG. 6. Schematic drawing of the application of a protease-regulatedantibody that cannot bind to two different antigens simultaneously.

FIG. 7. Schematic drawing of a monospecific protease-regulated antibody‘prodrug’ that can only bind antigen following protease activation toremove inactive blocking antibody domains (“Type 4”).

FIG. 8. Map of an expression vector for an IgG-like bispecific antibody.SignalP: signal peptide; VLam3E10: variable region of 3E10 lambda chain;Vk19G9: variable region of 19G9 kappa chain; C kappa: constant region ofkappa chain; DHFR: dihydrofolate reductase; V_(H): variable region ofheavy chain; Neo: neomycin resistant gene; 3E10VH: variable region of3E10 heavy chain; 19G9VH: variable region of 19G9 heavy chain; CH:constant region of heavy chain; Amp: ampicillin resistant gene.

FIG. 9. Map of expression vector of Fab-like bispecific antibody. LacZ,lac Z promoter; ompA and pho A, signal peptide; VL-link1-VK, variableregion of light chain of bispecific antibody against tissue factor andRG1; CL-kappa, constant region of kappa chain; VHs with linker, variableregion of heavy chain of bispecific antibody against tissue factor andRG1; CH1, the first constant region of IgG heavy chain; cat,chloramphenicol resistant gene.

FIG. 10. TF-binding ELISA. Four bispecific antibodies and parentalantibodies were analyzed for binding to TF. Antibodies were detectedwith HRP-conjugated anti-human IgG was used for detection. Curve fittingof the data was performed using a 4-parameter equation with the Solverfunction in Microsoft Excel. Positive control anti-TF IgG 3E10x:IC₅₀=2.0 nM (filled diamond, solid line); Linker 1 (SEQ ID NO: 1)IgG-like bispecific antibody: IC₅₀=0.78 nM (filled triangle, largedashed line); Linker 2 (SEQ ID NO: 2) IgG-like bispecific antibody:IC₅₀=0.93 nM (open triangle, small dashed line); Linker 3 (SEQ ID NO: 3)IgG-like bispecific antibody: IC₅₀=1.06 nM (filled square, alternatingsmall and large dashed line); Linker 4 (SEQ ID NO: 4) IgG-likebispecific antibody: IC₅₀=1.01 nM (open square, two large and one smalldashed line); and negative control anti-RG1 IgG 19G9: no binding (opendiamond, solid line).

FIG. 11. RG1-binding ELISA. A bispecific antibody containing Linker 1,anti-RG1 antibody 19G9, and polyclonal nonimmune control human IgG kappawere analyzed for binding to RG-1. Curve fitting of the data wasperformed using a 4-parameter equation with the Solver function inMicrosoft Excel. Positive control anti-RG1 IgG 19G9: IC₅₀=1.4 nM (filleddiamond, solid line); Linker 1 (SEQ ID NO: 1) IgG-like bispecificantibody: IC₅₀=1.1 nM (filled triangle, large dashed line); and negativecontrol nonimmune polyclonal human IgG kappa: no binding (open triangle,small dashed line).

FIG. 12. Measurement of the antigen-binding activity of a bispecificprotease-regulated antibody using a sandwich antigen-binding ELISA. Thelinker of this antibody contained cleavage sites for enterokinase.

FIG. 13. Measurement of the antigen-binding activity ofprotease-regulated antibodies 3E10-Type1-Fab and 19G9-Type1-Fab. Thecontrols are designated 3E10-Reg-Fab, 19G9-Reg-Fab, and HuFab.

FIG. 14. Measurement of the antigen-binding activity of Fab-likeprotease-regulated antibodies H1L1, H1L4, H1L7, H4L7, and H5L5 (Type 2)in the absence and presence of enterokinase. Parental antibodies 3E10and 19G9, and polyclonal human Fab were used as control.

FIG. 15. Measurement of the antigen-binding activity of Fab-likeprotease-regulated antibodies H2L1, H2L2, and H2L8 (Type 3) and H3L1,H3L4, and H5L4 (Type 4) in the absence and presence of enterokinase.Parental antibodies 3E10 and 19G9, and polyclonal human Fab were used ascontrol.

FIG. 16. Western blots of protease-regulated antibody 3E10-Type1-Fabdetected with anti-Myc antibody (A) or anti-kappa chain antibody (B).Lane 1 and 2: 3E10-Type1-Fab without or with enterokinase digestion,respectively. Lane 3 and 4: 3E10-Reg-Fab without or with enterokinasedigestion, respectively.

FIG. 17. Western blots of Fab-like protease-regulated antibodies H1L1,H1L7, and H5L5 (Type 2) in the absence and presence of enterokinase.Antibodies were detected with anti-IgG(H+L) antibody. Lane 1 and 2: H1L1without or with enterokinase digestion, respectively. Lane 3 and 4: H1L7without or with enterokinase digestion, respectively. Lane 5 and 6: H5L5without or with enterokinase digestion, respectively. Lane 7:3E10-Reg-Fab.

FIG. 18. Western blots of Fab-like protease-regulated antibodies H2L2and H2L8 (Type 3) and H3L4 (Type 4) in the absence and presence ofenterokinase. Antibodies were detected with anti-Myc antibody. Lane 1and 2: H2L2 without or with enterokinase digestion, respectively. Lane 3and 4: H2L8 without or with enterokinase digestion, respectively. Lane 5and 6: H3L4 without or with enterokinase digestion, respectively.

DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described and as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” is a reference to one or more antibodies and includesequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are hereby incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

“Antibody” as used herein includes intact immunoglobulin molecules(e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA), as well asfragments thereof, such as Fab, F(ab′)₂, scFv, Fv, and diabody which arecapable of specific binding to an epitope of a protein. The termantibody also extends to other protein scaffolds that are able to orientantibody complementarity-determining region (CDR) inserts into the sameactive binding conformation as that found in natural antibodies suchthat the binding to the target antigen observed with these chimericproteins is maintained relative to the binding activity of the naturalantibody from which the CDRs were derived.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules;monospecific antibodies; bispecific antibodies; and multispecificantibodies formed from antibody fragments.

The term “autoimmune diseases” includes, but is not limited to, multiplesclerosis, rheumatoid arthritis, lupus, type I diabetes mellitus,Crohn's disease, autoimmune hemolytic anemia, autoimmune hepatitis,glomerulonephritis, inflammatory bowel disease, myocarditis, psoriasis,thyroiditis, ulcerative colitis, and Graves'disease.

The terms “biological sample” or “patient sample” as used herein, refersto a sample obtained from an organism or from components (e.g., cells)of an organism. The sample may be of any biological tissue or fluid. Thesample may be a “clinical sample” which is a sample derived from apatient. Such samples include, but are not limited to, sputum, blood,serum, plasma, blood cells (e.g., white cells), tissue samples, biopsysamples, urine, peritoneal fluid, and pleural fluid, saliva, semen,breast exudate, cerebrospinal fluid, tears, mucous, lymph, cytosols,ascites, amniotic fluid, bladder washes, and bronchioalveolar lavages orcells therefrom, among other body fluid samples. The patient samples maybe fresh or frozen, and may be treated with heparin, citrate, or EDTA.Biological samples may also include sections of tissues such as frozensections taken for histological purposes.

The term “cancer” includes, but is not limited to, solid tumors, such ascancers of the breast, respiratory tract, brain, reproductive organs,digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid, and their distant metastases. The term alsoincludes lymphomas, sarcomas, and leukemias.

The term “conjugate” refers to an antibody chemically linked to achemical moiety, such as a therapeutic or cytotoxic agent.

The term “infectious diseases” includes, but is not limited to,HTV/AIDS, lower respiratory infections, diarrheal diseases,tuberculosis, malaria, measles, pertussis, tetanus, meningitis,syphilis, hepatitis B and tropical diseases.

The term “linker” refers to a peptide (or polypeptide) comprising two ormore amino acid residues joined by peptide bonds and used to link one ormore antibody domains. The linker may contain one or more proteasecleavage sites.

The term “protease” refers to any enzyme, including the endopeptidasesand exopeptidases, which catalyze the hydrolytic breakdown of proteinsinto peptides or amino acids.

The present invention is directed to the design and production ofmonospecific, bispecific antibodies, and multispecific antibodies. Forexample, the bispecific antibodies and multispecific antibodies maycomprise tandem linked V_(H)a-V_(H)b-V_(H)c . . . C_(H) in onepolypeptide and V_(L)a-V_(L)b-V_(L)c . . . C_(L) in another polypeptide.Alternately, the V_(H) and V_(L) domains may be exchanged from onepolypeptide to another to create polypeptides such asV_(H)a-V_(L)b-V_(H)c . . . C_(H) and V_(L)a-V_(H)b-V_(L)c . . . C_(L).The two polypeptides may form □immers in the Fab format or the halfIgG-like format, or two of each polypeptide may form a fourpolypeptide-containing homodimer of the IgG-like format. Thesebispecific or multispecific antibodies or antibody fragments thereof maysimultaneously bind different antigens or different epitopes of the sameantigen.

As an example, a recombinant IgG-like bispecific antibody may beconstructed by the tandem linking of two different V_(H) domains of aheavy chain and two different V_(L) domains of a light chain. Theconstruct is exemplified as follows:

-   -   heavy chain=NH₂-V_(H)1-V_(H)2-C_(H)1-C_(H)2-C_(H)3-COOH    -   light chain=NH₂-V_(L)1-V_(L)2-C_(L).

Another bispecific antibody may comprise the following:

-   -   heavy chain=NH₂-V_(L)1-V_(H)2-C_(H)1-C_(H)2-C_(H)3-COOH    -   light chain=NH₂-V_(H)1-V_(L)2-C_(L)-COOH.

The present invention also relates to protease-regulated antibodies.Protease-regulated antibodies may be, for example, monospecificantibodies, bispecific antibodies, multispecific antibodies, orantibodies with sequential binding-activity upon protease digestion ineither, for example, Fab-like or IgG-like format. Protease control orregulation may be provided by a selective protease site located in, forexample, a linker. These protease-regulated antibodies may be utilizedfor the diagnosis and treatment of various diseases including but notlimited to cancer, infectious disease, and autoimmune diseases, andprovide an additional level of control for biological drugs fortherapeutic or diagnostic applications.

Protease-regulated antibodies may comprise a heavy chain (H) variabledomain (V_(H))-linker-heavy chain constant domain (CH) in onepolypeptide and a light chain (L) variable domain (V_(L))-linker-lightchain constant domain (CL) in another polypeptide. Bispecificprotease-regulated antibodies may comprise, for example,V_(H1)-linker-V_(H2)-CH in one polypeptide and V_(L1)-linker-V_(L2)-CLin another polypeptide, both regulated by proteolytic cleavage of thelinker. Alternately, the V_(H) and V_(L) domains in the bispecificprotease-regulated antibodies may be exchanged from one polypeptide toanother polypeptide to create polypeptides such as, for example,V_(H1)-linker-V_(L2)-CH and V_(L1)-linker-V_(H2)-CL. The twopolypeptides may form □immers, for example, in a Fab-like format, ahalf-IgG-like format, or an IgG-like format (e.g., two of eachpolypeptide forming a four polypeptide-containing homodimer). Thebispecific and sequential protease-regulated antibodies or antibodyfragments may (1) simultaneously bind two different antigens ordifferent epitopes of the same antigen, (2) sequentially bind twodifferent antigens or different epitopes on the same antigen in a mannerthat may be dependent on the length, adjacent sequence, and design ofthe linker, or (3) a monospecific protease-activated binder which is inlatent or prodrug form prior to protease digestion and which is switchedon by protease cleavage. Libraries of bispecific protease-regulatedantibodies in the Fab-like format can be readily created, expressed inbacteria, and screened for specific functionalities, includingsusceptibility of the linker to cleavage by a specific protease andoptimization of this cleavage step.

Several types of protease-regulated antibodies are described hereinwhereby antibody formats are designed with selectivity due to specificprotease-dependent binding or protease-specific functionality. Inparticular, these protease-regulated antibodies may be described bydeletions and/or additions of antibody framework, location of thelinker, and its properties including length and solvent accessibility.Furthermore, the linker may contain a cleavage site specific for aprotease found in a target cell or tissue. One example of aprotease-regulated antibody may contain a protease site in a linkerlocated between the variable domain and the constant region domain andthis antibody may bind only one antigen as illustrated in FIG. 1 (“Type1”).

Another example of a protease-regulated antibody may simultaneously bindtwo different antigens or two different epitopes as shown in FIGS. 2 and3 (“Type 2”) in the absence of a protease. The first V_(H)/V_(L) domainsof this antibody bind to an antigen without blocking the secondV_(H)/V_(L) domains from binding to a second antigen. This antibody canbind to antigens without steric blocking of the CDR regions of thesecond variable domains. The simultaneous binding of thisprotease-regulated antibody to both antigens is prevented by proteolyticcleavage. That is, when the linker is cleaved by a protease, theantibody can only bind to the second antigen or separately bind twoantigens. Simultaneous antigen binding is important for antibodyfunction, for example, in cross-linking receptors, which may beprevented by proteolytic cleavage. Thus, an additional degree ofspecificity is added by including a protease site in the linker.

This bispecific protease-regulated antibody is more selective than amonospecific antibody because this antibody will specifically targetcells or tissues expressing both antigens. The additional degree ofspecificity is provided by the specific protease site in the linker. InFIG. 4, Cell A and Cell B express both Antigen 1 and Antigen 2, howeveronly Cell B expresses the selective protease. The bispecificprotease-regulated antibody with the uncleavable linker (i.e., thislinker does not contain the protease cleavage site) will bind to Cell Awith greater avidity because the bispecific antibody is able to bind toboth antigens. In contrast, the bispecific protease-regulated antibodywill bind to Cell B with lower avidity because the Antigen 1 bindingdomain is removed by proteolytic cleavage of the linker by the selectiveprotease expressed by Cell B (alternately, the selective protease may beexpressed by adjacent cells localized in the same tissue as Cell B).

In contrast, FIG. 5 illustrates a protease-regulated antibody that maysequentially bind to each antigen in a protease-dependent manner. Thatis, prior to protease cleavage of the linker, the protease-regulatedantibody binds to a first antigen and following protease cleavage, theantibody binds to a second antigen (“Type 3”). The V_(H)/V_(L) domainsof the N-terminal antibody bind to an antigen, but block the CDR regionsof the downstream V_(H)/V_(L) domains from binding to a second antigen.Protease cleavage of the linker allows removal of the N-terminalantibody, and removing the N-terminal antibody domains then permitsbinding to a second antigen. This allows for greater cell and/or tissueselectivity by requiring sequential binding.

In FIG. 6, Cell A and Cell B express both Antigen 1 and Antigen 2, butonly Cell B expresses the selective protease. In addition, Antigen 2 isa cell surface receptor that internalizes into the cell and allowsinternalization of antibodies that bind to it. The protease-regulatedantibody will bind to Antigen 1 expressed by Cell A and Cell B. However,only Cell B expresses the selective protease (or possibly cells adjacentto Cell B in the same tissue). The protease-regulated antibody will beactivated by proteolytic cleavage and internalized via Antigen 2expressed on Cell B. Thus, this protease-regulated antibody will bespecifically internalized by cells expressing Antigen 1, Antigen 2, andthe selective protease.

In an additional example, a protease-regulated antibody may not bind toan antigen before protease digestion, but may bind to antigen followingprotease digestion (“Type 4”). An example of this antibody isillustrated in FIG. 7. This monospecific protease-regulated antibodyalso contains a protease cleavage linker that allows removal of theN-terminal non-functional antibody which then leads to binding to anantigen by the functional antibody domains that are thus exposed. Type 4protease-regulated antibodies may be created by three approaches. In thefirst approach the protease cleavable linker sequence is modified sothat it prevents the N-terminal V_(H) and V_(L) domains of a Type IIIantibody (V_(H)1 and V_(L)1) from binding to the first antigen. Examplesof these linkers are shown in the sequences in Table 8. In the secondapproach, the linkers utilized in Type III antibodies shown in Tables 6and 7 are now combined with heterodimeric N-terminal V_(H) and V_(L)domains that have been mutated to destroy their antigen bindingfunction. Examples of this approach are shown in the sequences in Table9 in which the CDR3 of V_(H)1 and CDR3 of V_(L)1 are replaced by apoly-alanine sequence of a similar length as the respective CDR. In thethird approach, the linkers utilized in Type III antibodies shown inTables 6 and 7 are combined with homodimeric N-terminal domains derivedfrom the constant regions of antibodies. For example, the completeV_(H)1 and V_(L)1 domains of a Type III antibody are both replaced bythe same constant domain that is capable of heterodimerization, forexample, the CH3 domain of IgG or the CH4 domain of IgE.

These protease-regulated antibodies may be modified by protease cleavageof the linker as described below. For example, the protease-regulatedantibody illustrated in FIG. 1 (Type 1) contains a protease site in thelinker between the antigen binding domains and the Fc domain. Thisantibody will specifically target cells or tissues that present theantigen. When the linker is cleaved by the protease, the resultantprotease-regulated antibody releases the functional Fc portion. Intissues where the protease is present, this antibody will release the Fcportion which is essential to antigen crosslinking, and induce an immuneresponse such as antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). To illustrate, hepsin, a serineprotease, is expressed in both tumor tissue and normal liver tissue. Ina cancer patient treated with a protease-regulated antibody againsthepsin, the antibody would attack the tumor cells via the Fcportion-induced ADCC and CDC. However, in the liver, theprotease-regulated antibody would initially bind to hepsin, but the Fcportion would be cleaved by a liver-specific protease prior toinitiation of ADCC or CDC preventing liver toxicity.

The peptide (or polypeptide) linker of the protease-regulated antibodymay comprise two or more amino acid residues and may contain one or moreprotease cleavage sites. The linkers may alter antibody conformation,stability, and antigen-binding activities. The length of linkers mayrange, for example, from 0 to about 100 amino acid residues. Thefollowing are examples of linkers:

Linker 1: SDDDDK (SEQ ID NO: 1) Linker 2: GGGGSDDDDK (SEQ ID NO: 2)Linker 3: GGGGSDDDDKGGGGS (SEQ ID NO: 3) Linker 4: GGGGSGGGGSGGGGS(SEQ ID NO: 4) Linker 5: IHPVLSGLSRIVNGEDAVPG (SEQ ID NO: 5) Linker 6:VAAPFDDDDKIVGGYICEEN (SEQ ID NO: 6) Linker 7: ELLESYIDGRIVEGSDAEIG(SEQ ID NO: 7) Linker 8: STQSFNDFTRVVGGEDAKPG (SEQ ID NO: 8) Linker 9:PERGDNNLTRIVGGQECKDG (SEQ ID NO: 9) Linker 10: EDQEDQVDPRLIDGKMTRRG(SEQ ID NO: 10) Linker 11: KRNASKPQGRIVGGKVCPKG (SEQ ID NO: 11)Linker 12: SVCTTKTSTRIVGGTNSSWG (SEQ ID NO: 12) Linker 13: SRIVG(SEQ ID NO: 13) Linker 14: GSLVSGSCSQIINGEDCSPH (SEQ ID NO: 14)Linker 15: SRIIN (SEQ ID NO: 15) Linker 16: NKLVH (SEQ ID NO: 16)Linker 17: DKIID (SEQ ID NO: 17) Linker 18: FNVLG (SEQ ID NO: 18)Linker 19: TRAIG (SEQ ID NO: 19) Linker 20: TRLDP (SEQ ID NO: 20)Linker 21: TRIIK (SEQ ID NO: 21) Linker 22: SGSNQ (SEQ ID NO: 22)Linker 23: SKVLN (SEQ ID NO: 23) Linker 24: NKIIG (SEQ ID NO: 24)Linker 25: DKLLE (SEQ ID NO: 25)

Table 1 illustrates the excision site of several proteases.

TABLE 1 Cleavage Enzyme/ Excision site ↓ Self-CleavageAsp-Asp-Asp-Asp-Lys↓ Enterokinase (DDDDK) (SEQ ID NO: 26)Ile-Glu/Asp-Gly-Arg↓ Factor Xa  (IE/DGR) protease (SEQ ID NO: 27)Leu-Val-Pro-Arg↓Gly-Ser Thrombin (LVPR | GS) (SEQ ID NO: 28)Glu-Asn-Leu-Tyr-Phe-Gln↓Gly TEV protease (ENLYFQ | G) (SEQ ID NO: 29)Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro Human rhinovirus (LEVLFQ | GP)3C protease (SEQ ID NO: 30) Ser-Ser-Val-Phe-Ala-Gln↓Ser-Ile-PCSK9 (NARC-1) Pro (SSVFAQ | SIP) (SEQ ID NO: 31)Lys-Gln-Leu-Arg↓Val-Val-Asn-Gly Hepsin (KQLR | VVNG) (SEQ ID NO: 32)Specific intein-encoded Intein 1 & sequences intein 2 Signal sequencesSignal peptidases

The cleavage sites of additional proteases that may be incorporated in alinker are described in Table 2.

TABLE 2 TUMOR ASSOCIATED PROTEASES (Extracellular Or Intracellular) ADAMmetallopeptidase domain 9 (meltrin gamma) ADAM metallopeptidase domain10 ADAM metallopeptidase domain 17 (TNFalpha, converting enzyme) ADAMmetallopeptidase domain 28 ADAM-like, decysin 1 ADAM metallopeptidase,thrombospondin type 1 motif 1 ADAM metallopeptidase, thrombospondin type1 motif 5, aggrecanase-2 ADAMTS-like 3 ADAMTS-like 4 Beta-siteAPP-cleaving enzyme 1 Bleomycin hydrolase Bone morphogenetic protein 1Complement component 1, r subcomponent Complement component 1, ssubcomponent Calpain 2, (m/II) large subunit Caspase 1,apoptosis-related cysteine peptidase (IL-1β convertase) Caspase 2,apoptosis-related cysteine peptidase Caspase 3, apoptosis-relatedcysteine peptidase Caspase 4, apoptosis-related cysteine peptidaseCaspase 6, apoptosis-related cysteine peptidase Caspase 7,apoptosis-related cysteine peptidase Caspase 9, apoptosis-relatedcysteine peptidase Complement factor D (adipsin) CASP8 and FADD-likeapoptosis regulator Cathepsin B Cathepsin F Cathepsin H Cathepsin KCathepsin L Cathepsin L2 Cathepsin O Cathepsin S Cylindromatosis (turbantumor syndrome) Extra spindle pole bodies homolog 1 (S. Cerevisiae)Granzyme A (granzyme 1, CTL-associated serine esterase 3)Histocompatibility (minor) 13 Hepsin (transmembrane protease, serine 1)HtrA serine peptidase 1 Kallikrein-related peptidase 11 Legumain Lonpeptidase 1, mitochondrial Mucosa associated lymphoid tissue lymphomatranslocation gene 1 Membrane-bound transcription factor peptidase, site1 Matrix metallopeptidase 1 (interstitial collagenase) Matrixmetallopeptidase 12 (macrophage elastase) Matrix metallopeptidase 14(membrane-inserted) Matrix metallopeptidase 9 (gelatinase B, 92 kDa typeIV collagenase) N-acetylated alpha-linked acidic dipeptidase-like 1Napsin A aspartic peptidase Pregnancy-associated plasma protein A,pappalysin 1 Proprotein convertase subtilisin/kexin type 5 Plasminogenactivator, tissue Plasminogen activator, urokinase Peptidase(mitochondrial processing) beta Protease, serine, 3 (mesotrypsin)Protease, serine, 8 (prostasin) Proteasome (prosome, macropain) subunit,alpha type, 1 Proteasome (prosome, macropain) subunit, alpha type, 6Proteasome (prosome, macropain) subunit, beta type, 4 Proteasome(prosome, macropain) subunit, beta type, 9 Proteasome (prosome,macropain) subunit, beta type, 10 SUMO1/sentrin specific peptidase 1Suppression of tumorigenicity 14 (colon carcinoma) Tubulointerstitialnephritis antigen Torsin family 1, member A (torsin A) Tripeptidylpeptidase I Tripeptidyl peptidase II Tryptase alpha/beta 1 Tryptasealpha/beta 1 Ubiquitin specific peptidase 4 (proto-oncogene) Ubiquitinspecific peptidase 10 Ubiquitin specific peptidase 11 Ubiquitin specificpeptidase 14 (tRNA-guanine transglycosylase) Ubiquitin specificpeptidase 15 Ubiquitin specific peptidase 16 Ubiquitin specificpeptidase 18 Ubiquitin specific peptidase 25 YME1-like 1 (S. cerevisiae)Zinc metallopeptidase (STE24 homolog, yeast)

The protease-regulated antibodies of the present invention may bind oneor more antigens. These antigens may be selected from the groupconsisting of cytokines, cell surface receptors, enzymes, and receptors.These antigens include, but are not limited to, CD3, CD4, CD8, CD20,CD25, CD28, CD33, CD52, IL-2, IL-7, IL-8, TNF-alpha, TGF-beta INF-beta,INF-gamma, GMCSF, GCSF, VEGF, C5, EpCAM, EGF receptor, CD2 receptor, IL2 receptor, IgE receptor, intergrin, and MHC class II.

The antibodies of the present invention may be utilized for thediagnosis and therapy of various diseases. For example, antibodiesdirected against human immunological cells and tumor-associated antigenmay be used for cancer therapy. These antibodies may also be directedagainst tumor-associated antigen and toxic agents or enzymes for use asa cancer therapeutic. The antibodies of the present invention may alsobe utilized for the treatment of hemophilia and thrombosis as well asstem cell transplantation. These antibodies may be used for theselective stimulation and expansion of lymphocyte subset. In addition,these antibodies may used for the detection of disease-related antigens.

For cancer immunotherapy, bispecific antibodies may be used to recruitthe immune system to attach tumor cells. Targets on immunological cellsinclude, but are not limited to, CD3, CD8, and Fc receptor.Tumor-associated antigens include, but are not limited to, Her2, EGFreceptor, CD20, CA-125, and carcinoembryonic antigen (CEA). For example,a bispecific antibody against CD8 and Her2 can direct CD8-expressingcytotoxic lymphocytes to attack Her2 expressing breast cancer cells.

The antibodies or antibody fragments of the invention, or compositionsincluding the antibodies or fragments, can include a cytoxic agent thatis conjugated to the antibody or fragment. In one aspect, the cytotoxicagent is monomethylauristatin-E (MMAE), however, other cytoxic agentsare also provided, which can include, for example, functional analogs ofMMAE (e.g. monomethylauristatin-F), and other cytotoxic agents, e.g.,aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-I 1), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactiπomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FUdR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribomiclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin, orcombinations thereof. Any of the cytoxic agents can also includefunctional analogs thereof.

Antibody Technology

A number of technologies are available to produce antibodies. Forexample, phage-antibody technology may be used to generate antibodies(Knappik, et al., J. Mol. Biol. 296:57-86, 2000). Another approach forobtaining antibodies is to screen a DNA library from B cells asdescribed by Dower, et al., (WO 91/17271) and McCafferty, et al., (WO92/01047). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies are selected by affinity enrichment for binding to a selectedprotein. Antibodies may also be produced using trioma methodology(Oestberg, et al., Hybridoma 2:361-367, 1983; U.S. Pat. No. 4,634,664;U.S. Pat. No. 4,634,666).

Antibodies may also be purified from any cell that expresses theantibodies, including host cells that have been transfected withantibody-encoding expression constructs. The host cells may be culturedunder conditions whereby the antibodies are expressed. Purified antibodymay be separated from other cellular components that may associate withthe antibody in the cell, such as certain proteins, carbohydrates, orlipids using methods well known in the art. Such methods include, butare not limited to, size exclusion chromatography, ammonium sulfatefractionation, ion exchange chromatography, affinity chromatography, andpreparative gel electrophoresis. Purity of the preparations may beassessed by any means known in the art, such as SDS-polyacrylamide gelelectrophoresis. A preparation of purified antibodies may contain morethan one type of antibody.

Alternatively, antibodies may be produced using chemical methods tosynthesize its amino acid sequence, such as by direct peptide synthesisusing solid-phase techniques (see, e.g., Merrifield, J. Am. Chem. Soc.85:2149-2154, 1963; Roberge, et al., Science 269:202-204, 1995). Proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally,fragments of antibodies may be separately synthesized and combined usingchemical methods to produce a full-length molecule.

The antibodies of the present invention may be generated from parentalantibodies. Parent antibodies may be selected from various antibodiescapable of binding specific targets and well known in the art, such as,but not limited to, but are not limited to anti-TNF antibody, anti-IL-12antibody; anti-IL-18 antibody, anti-05, anti-CD147, anti-gp120,anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti-ICAM-1, anti-CD2,anti-EGFR, anti-TGF-beta 2, anti-E-selectin, anti-Her2/neu, anti-CD14,anti-ICAM-3, anti-CD80, anti-CD4, anti-CD3, anti-CD23,anti-beta2-integrin, anti-CD52, anti-CD22, anti-CD20, anti-CD25,anti-CD33, anti-HLA, anti-IL-1alpha, anti-IL-1, anti-IL-1 receptor,anti-IL-2 receptor, anti-IL-4, anti-IL4 receptor, anti-IL5, anti-IL-5receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13receptor, anti-IL-17, and anti-IL-23. Parent antibodies may also beselected from various therapeutic antibodies including, but are notlimited to, rituximab, trastuzumab, pertuzumab, cetuximab, alemtuzumab,muromonab, ibritumomab, gemtuzumab ozogamicin, alefacept, abciximab,basiliximab, palivizumab, infliximab, adalimumab, etanercept,natalizumab, bevacizumab, omalizumab, efalizumab, clenoliximab,labetuzumab, epratuzumab, and visilizumab.

The newly synthesized molecules may be substantially purified bypreparative high performance liquid chromatography (see, e.g.,Creighton, Proteins: Structures and Molecular Principles, WH Freeman andCo., New York, N.Y., 1983). The composition of a synthetic polypeptidemay be confirmed by amino acid analysis or sequencing (e.g., using Edmandegradation).

The present invention also relates to bispecific or bifunctionalantibodies that have one binding site that specifically binds to a firstantigen and a second binding site that specifically binds to a secondantigen. This results in multi-functional valency, that is, an abilityto bind at least two different epitopes simultaneously.

Polynucleotides Encoding Antibodies

The present invention also relates to polynucleotides encodingantibodies. These polynucleotides may be used, for example, to producequantities of the antibodies for therapeutic or diagnostic use.

Polynucleotides of the present invention may also be isolated from hostcells, free of other cellular components such as membrane components,proteins, and lipids. Polynucleotides may be isolated using standardnucleic acid purification techniques, or synthesized using anamplification technique such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide may be used to obtain isolated polynucleotides encodingantibodies of the invention. For example, restriction enzymes and probesmay be used to isolate polynucleotides which encode antibodies.

Antibody-encoding cDNA molecules may be made with standard molecularbiology techniques, using mRNA as a template. Thereafter, cDNA moleculesmay be replicated using molecular biology techniques known in the artand disclosed in manuals such as Sambrook, et al., (Molecular Cloning: ALaboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press;Cold Spring Harbor, N.Y.; 1989) Vol. 1-3). An amplification technique,such as PCR, may be used to obtain additional copies of thepolynucleotides. Alternatively, synthetic chemistry techniques may beused to synthesize polynucleotides encoding antibodies of the invention.

To express a polynucleotide encoding an antibody, the polynucleotide maybe inserted into an expression vector that contains the necessaryelements for the transcription and translation of the inserted codingsequence. Methods that are well known to those skilled in the art may beused to construct expression vectors containing sequences encodingantibodies and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described, for example, in Sambrook, et al. (1989) and in Ausubel,et al., (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1995).

A variety of expression vector/host systems may be utilized to containand express sequences encoding antibodies. These include, but are notlimited to, microorganisms, such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV); or bacterial expressionvectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in strength andspecificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters can be used. Thebaculovirus polyhedrin promoter may be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses may be used. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding an antibody,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

General texts describing additional molecular biological techniquesuseful herein, including the preparation of antibodies include Bergerand Kimmel (Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al.,(Molecular Cloning: A Laboratory Manual, (Second Edition, Cold SpringHarbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3);Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.],Current Protocols, a joint venture between Green Publishing Associates,Inc. and John Wiley & Sons, Inc. (supplemented through 2000)); Harlow etal., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988), Paul [Ed.]); Fundamental Immunology,(Lippincott Williams & Wilkins (1998)); and Harlow, et al., (UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1998)).

Assays

The affinity (K_(d)) of antibody binding to an antigen may be assayedusing any method known in the art including, for example, immunoassayssuch as enzyme-linked immununospecific assay (ELISA), BimolecularInteraction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky; Anal.Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol.5:699-705, 1995), and fluorescence-activated cell sorting (FACS) forquantification of antibody binding to cells that express an antigen. BIAis a technology for analyzing biospecific interactions in real time,without labeling any of the interactants (e.g., BIAcore™). Changes inthe optical phenomenon surface plasmon resonance (SPR) may be used as anindication of real-time reactions between biological molecules.

The present invention also relates to the use of quantitativeimmunoassays to measure levels of proteins in patient samples. Manyformats may be adapted for use with the methods of the presentinvention. For example, the detection and quantitation of a protein inpatient samples may be performed, by enzyme-linked immunosorbent assays,radioimmunoassays, dual antibody sandwich assays, agglutination assays,fluorescent immunoassays, immunoelectron and scanning microscopy, amongother assays commonly known in the art. The quantitation of a protein insuch assays may be adapted by conventional methods known in the art.Serial changes in circulating a protein levels may be detected andquantified by a sandwich assay in which the capture antibody has beenimmobilized using conventional techniques on the surface of the support.

Suitable supports include, for example, synthetic polymer supports, suchas polypropylene, polystyrene, substituted polystyrene, polyacrylamides(such as polyamides and polyvinylchloride), glass beads, agarose, andnitrocellulose.

The antibodies useful to identify proteins may be labeled in anyconventional manner. An example of a label is horseradish peroxidase,and an example of a method of labeling antibodies is by usingbiotin-strepavidin complexes.

As appropriate, antibodies used in the immunoassays of this inventionthat are used as tracers may be labeled in any manner, directly orindirectly, that results in a signal that is visible or can be renderedvisible. Detectable marker substances include radionuclides, such as ³H,¹²⁵I, and ¹³¹I; fluorescers, such as, fluorescein isothiocyanate andother fluorochromes, phycobiliproteins, phycoerythin, rare earthchelates, Texas red, dansyl and rhodamine; colorimetric reagents(chromogens); electron-opaque materials, such as colloidal gold;bioluminescers; chemiluminescers; dyes; enzymes, such as, horseradishperoxidase, alkaline phosphatases, glucose oxidase, glucose-6-phosphatedehydrogenase, acetylcholinesterase, alpha beta-galactosidase, amongothers; coenzymes; enzyme substrates; enzyme cofactors; enzymeinhibitors; enzyme subunits; metal ions; free radicals; or any otherimmunologically active or inert substance which provides a means ofdetecting or measuring the presence or amount of immunocomplex formed.Exemplary of enzyme substrate combinations are horseradish peroxidaseand tetramethyl benzidine (TMB), and alkaline phosphatases andparanitrophenyl phosphate (pNPP).

Another detection and quantitation systems according to this inventionproduce luminescent signals, bioluminescent (BL) or chemiluminescent(CL). In chemiluminescent (CL) or bioluminescent (BL) assays, theintensity or the total light emission is measured and related to theconcentration of the unknown analyte. Light can be measuredquantitatively using a luminometer (photomultiplier tube as thedetector) or charge-coupled device, or qualitatively by means ofphotographic or X-ray film. The main advantages of using such assays istheir simplicity and analytical sensitivity, enabling the detectionand/or quantitation of very small amounts of analyte.

Exemplary luminescent labels are acridinium esters, acridinium sulfonylcarboxamides, luminol, umbelliferone, isoluminol derivatives,photoproteins, such as aequorin, and luciferases from fireflies, marinebacteria, Vargulla and Renilla. Luminol can be used optionally with anenhancer molecule such as 4-iodophenol or 4-hydroxy-cinnamic acid.Typically, a CL signal is generated by treatment with an oxidant underbasic conditions.

Additional luminescent detection systems are those wherein the signal(detectable marker) is produced by an enzymatic reaction upon asubstrate. CL and BL detection schemes have been developed for assayingalkaline phosphatases (AP), glucose oxidase, glucose 6-phosphatedehydrogenase, horseradish peroxidase (HRP), and xanthine-oxidaselabels, among others. AP and HRP are two enzyme labels which can bequantitated by a range of CL and BL reactions. For example, AP can beused with a substrate, such as an adamantyl 1,2-dioxetane aryl phosphatesubstrate (e.g. AMPPD or CSPD; Kricka, L. J., “Chemiluminescence andBioluminescence, Analysis by,” Molecular Biology and Biotechnology: AComprehensive Desk Reference (ed. R. A. Meyers) (VCH Publishers; N.Y.,N.Y.; 1995)); for example, a disodium salt of4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2′-adamantane],with or without an enhancer molecule such as 1-(trioctylphosphoniummethyl)-4-(tributylphosphonium methyl)benzene diochloride. HRP is may beused with substrates, such as,2′,3′,6′-trifluorophenyl-methoxy-10-methylacridan-9-carboxylate.

CL and BL reactions may be adapted for analysis not only of enzymes, butalso of other substrates, cofactors, inhibitors, metal ions, and thelike. For example, luminol, firefly luciferase, and marine bacterialluciferase reactions are indicator reactions for the production orconsumption of peroxide, ATP, and NADPH, respectively. They may becoupled to other reactions involving oxidases, kinases, anddehydrogenases, and may be used to measure any component of the coupledreaction (enzyme, substrate, cofactor).

The detectable marker may be directly or indirectly linked to anantibody used in an assay of this invention. Exemplary of an indirectlinkage of the detectable label is the use of a binding pair between anantibody and a marker or the use of a signal amplification system.

Examples of binding pairs that may be used to link antibodies todetectable markers are biotin/avidin, streptavidin, or anti-biotin;avidin/anti-avidin; thyroxine/thyroxine-binding globulin;antigen/antibody; antibody/anti-antibody; carbohydrate/lectins;hapten/anti-hapten antibody; dyes and hydrophobic molecules/hydrophobicprotein binding sites; enzyme inhibitor, coenzyme or cofactor/enzyme;polynucleic acid/homologous polynucleic acid sequence;fluorescein/anti-fluorescein; dinitrophenol/anti-dinitrophenol; vitaminB 12/intrinsic factor; cortisone, cortisol/cortisol binding protein; andligands for specific receptor protein/membrane associated specificreceptor proteins.

Various means for linking labels directly or indirectly to antibodiesare known in the art. For example, labels may be bound either covalentlyor non-covalently. Exemplary antibody conjugation methods are describedin Avarmeas, et al., Scan. J. Immunol. 8(Suppl. 7): 7, 1978); Bayer, etal., Meth. Enzymol. 62:308, 1979; Chandler, et al., J. Immunol. Meth.53:187, 1982; Ekeke and Abuknesha, J. Steroid Biochem. 11:1579, 1979;Engvall and Perlmann, J. Immunol. 109:129, 1972; Geoghegan, et al.,Immunol. Comm. 7:1, 1978; and Wilson and Nakane, Immunofluorescence andRelated Techniques, Elsevier/North Holland Biomedical Press; Amsterdam(1978).

Depending upon the nature of the label, various techniques may beemployed for detecting and quantitating the label. For fluorescers, alarge number of fluorometers are available. For chemiluminescers,luminometers or films are available. With enzymes, a fluorescent,chemiluminescent, or colored product may be determined or measuredfluorometrically, luminometrically, spectrophotometrically, or visually.

Various types of chemiluminescent compounds having an acridinium,benzacridinium, or acridan type of heterocyclic ring systems are otherexamples of labels. Examples of acridinium esters include thosecompounds having heterocyclic rings or ring systems that contain theheteroatom in a positive oxidation state including such ring systems asacridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, abenzimidazole cation, quinolinium, isoquinolinium, quinolizinium, acyclic substituted quinolinium, phenanthridinium, and quinoxalinium.

The tracer may be prepared by attaching to the selected antibody eitherdirectly or indirectly a reactive functional group present on theacridinium or benzacridinium ester, as is well known to those skilled inthe art (see, e.g., Weeks, et al., Clin. Chem. 29(8):1474-1479, 1983).Examples of compounds are acridinium and benzacridinium esters with anaryl ring leaving group and the reactive functional group present ineither the para or the meta position of the aryl ring. (see, e.g., U.S.Pat. No. 4,745,181 and WO 94/21823).

Methods of Use

As used herein, various terms are defined below.

The term “treatment” includes any process, action, application, therapy,or the like, wherein a subject (or patient), including a human being, isprovided medical aid with the object of improving the subject'scondition, directly or indirectly, or slowing the progression of acondition or disorder in the subject.

The term “combination therapy” or “co-therapy” means the administrationof two or more therapeutic agents to treat a disease, condition, and/ordisorder. Such administration encompasses co-administration of two ormore therapeutic agents in a substantially simultaneous manner, such asin a single capsule having a fixed ratio of active ingredients or inmultiple, separate capsules for each inhibitor agent. In addition, suchadministration encompasses use of each type of therapeutic agent in asequential manner.

The antibodies of the invention may be administered in combination withthe following agents: cytotoxic agent, angiogenesis inhibitors,antirheumatic agent, muscle relaxant, narcotic, non-steroidanti-inflammatory drug, analgesic, anesthetic, sedative, localanesthetic, neuromuscular blocker, antimicrobial agent, immunoglobulins,antidepressant, asthma medication, cytokine, and cytokine antagonist.

For example, the antibodies of the invention may be administered incombination with various anti-cancer agents including, but not limitedto, bleomycin, docetaxel, doxorubicin, edatrexate, erlotinib, etoposide,finasteride, flutamide, gemcitabine, genitinib, goserelin acetate,granisetron, imatinib, irinotecan, ondansetron, paclitaxel,pegaspargase, pilocarpine hydrochloride, porfimer sodium, interleukin-2,rituximab, topotecan, trastuzumab, triapine, vincristine, andvinorelbine tartrate, or therapeutic antibodies or fragments thereof, oranti-angiogenic agent, such as, for example, angiostatin, bevacizumab,sorafenib, baculostatin, canstatin, maspin, anti-VEGF antibodies orpeptides, anti-placental growth factor antibodies or peptides,anti-Flk-1 antibodies, anti-Fit-1 antibodies or peptides, lamininpeptides, fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin 12, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM1O1, Marimastat, pentosan polysulphate,angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16Kprolactin fragment, Linomide, thalidomide, pentoxifylline, genistein,TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline.

The phrase “therapeutically effective” means the amount of each agentadministered that will achieve the goal of improvement in a disease,condition, and/or disorder severity, while avoiding or minimizingadverse side effects associated with the given therapeutic treatment.

The term “pharmaceutically acceptable” means that the subject item isappropriate for use in a pharmaceutical product.

The antibodies of this invention are expected to be valuable astherapeutic agents. Accordingly, an embodiment of this inventionincludes a method of treating the various conditions in a patient(including mammals) which comprises administering to said patient acomposition containing an amount of an antibody of the invention that iseffective in treating the target condition.

The antibodies of the present invention may be used in the treatment orprevention of various diseases including, but not limited to, cancer,infectious disease, and autoimmune diseases.

The antibodies of the present invention or compositions including theantibodies may include a cytotoxic agent (e.g., monomethylauristatin-E)that is conjugated to the antibody.

Antibodies of the present invention may be administered alone or incombination with one or more additional therapeutic agents. Combinationtherapy includes administration of a single pharmaceutical dosageformulation which contains an antibody of the present invention and oneor more additional therapeutic agents, as well as administration of theantibody of the present invention and each additional therapeutic agentsin its own separate pharmaceutical dosage formulation. For example, anantibody of the present invention and a therapeutic agent may beadministered to the patient together in a single oral dosage compositionor each agent may be administered in separate oral dosage formulations.

Where separate dosage formulations are used, the antibody of the presentinvention and one or more additional therapeutic agents may beadministered at essentially the same time (e.g., concurrently) or atseparately staggered times (e.g., sequentially).

To assess the ability of a particular antibody to be therapeuticallyuseful to treat cancer, as an example, the antibody may be tested invivo in a mouse xenograft tumor model. An example of a therapeutic modelis detailed in Example 8.

Pharmaceutical Compositions

The antibodies described herein may be provided in a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier may be non-pyrogenic. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier including, but notlimited to, saline, buffered saline, dextrose, and water. A variety ofaqueous carriers may be employed including, but not limited to saline,glycine, or the like. These solutions are sterile and generally free ofparticulate matter. These solutions may be sterilized by conventional,well known sterilization techniques (e.g., filtration). The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions such as pH adjusting andbuffering agents, and the like. The concentration of the antibody of theinvention in such pharmaceutical formulation may vary widely, and may beselected primarily based on fluid volumes, viscosities, etc., accordingto the particular mode of administration selected. If desired, more thanone type of antibody may be included in a pharmaceutical composition.

The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. In addition to theactive ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations which may be used pharmaceutically. Pharmaceuticalcompositions of the invention may be administered by any number ofroutes including, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, parenteral,topical, sublingual, or rectal means.

Formulations suitable for subcutaneous, intravenous, intramuscular, andthe like; suitable pharmaceutical carriers; and techniques forformulation and administration may be prepared by any of the methodswell known in the art (see, e.g., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 20^(th) edition, 2000).

Diagnostic Methods

The present invention also provides diagnostic methods with which aparticular antigen may be detected in a patient sample or biologicalsample. Such diagnostic methods may be used, for example, to diagnosedisorders in which a particular antigen is elevated or reduced. Suchdisorders include, but are not limited to, cancer, infectious disease,and autoimmune diseases. As an example, when used for diagnosis,detection of an amount of the antibody-antigen complex in a sample froma patient which is greater than an amount of the complex in a normalsample identifies the patient as likely to have the disorder

The patient sample may be contacted with an antibody of the invention,and the patient sample may then be assayed for the presence of anantibody-antigen complex. As described above, the antibody may comprisea detectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic label, such as horseradish peroxidase,alkaline phosphatase, or luciferase.

Optionally, the antibody may be bound to a solid support, which mayaccommodate automation of the assay. Suitable solid supports include,but are not limited to, glass or plastic slides, tissue culture plates,microtiter wells, tubes, silicon chips, or particles such as beads(including, but not limited to, latex, polystyrene, or glass beads). Anymethod known in the art may be used to attach the antibody to the solidsupport, including use of covalent and non-covalent linkages, passiveabsorption, or pairs of binding moieties attached to the antibody andthe solid support. Binding of antigen and the antibody may beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicrocentrifuge tubes.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to the amount of an antibody that may be used to effectivelytreat a disease (e.g., cancer) compared with the efficacy that isevident in the absence of the therapeutically effective dose.

The therapeutically effective dose may be estimated initially in animalmodels (e.g., rats, mice, rabbits, dogs, or pigs). The animal model mayalso be used to determine the appropriate concentration range and routeof administration. Such information may then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity (e.g., ED₅₀—the dose therapeuticallyeffective in 50% of the population and LD₅₀—the dose lethal to 50% ofthe population) of an antibody may be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit may be expressed as the ratio, LD₅₀/ED₅₀. The data obtained fromanimal studies may used in formulating a range of dosage for human use.The dosage contained in such compositions may be within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage may be determined by the practitioner, in light offactors related to the patient who requires treatment. Dosage andadministration may be adjusted to provide sufficient levels of theantibody or to maintain the desired effect. Factors that may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Effective in vivodosages of an antibody are in the range of about 5 μg to about 500 μg/kgof patient body weight.

The mode of administration of antibody-containing pharmaceuticalcompositions of the present invention may be any suitable route whichdelivers the antibody to the host. As an example, pharmaceuticalcompositions of the invention may be useful for parenteraladministration (e.g., subcutaneous, intramuscular, intravenous, orintranasal administration).

All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples, whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLES

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only, and are not to be construed as limiting the scope ofthe invention in any manner. All publications mentioned herein areincorporated by reference in their entirety.

Example 1 Construction, Expression, and Purification of Fab-LikeAntibody

Antibodies 3E10 and 19G9 recognize tissue factor (TF) andtumor-associated antigen RG1, respectively. These two antibodies wereused to construct protease-regulated antibodies containing the proteasesite DDDDK (SEQ ID NO: 26) linker located between the antigen bindingdomains and constant region domain. Specifically, these antibodiescontained V_(L)-DDDDK-CL on the light chain and V_(H)-DDDDK-CH1-Myc-His6on the heavy chain, where the linker is cleavable by enterokinase, andMyc and His6 are tags for detection and purification. The DNA sequencesfor the two antibodies were cloned into bacterial expression vectorsusing standard molecular biology technologies, and the constructs wereconfirmed by DNA sequencing. Examples of plasmid are shown in FIGS. 8and 9. The plasmid containing either 3E10 or 19G9 was expressed andpurified from bacterial strain TG1. Briefly, a single colony of bacteriastrain TG1 containing the antibody expression plasmid was selected andgrown overnight in 8 ml of 2× YT medium in the presence of 34 μg/mlchloramphenicol and 1% glucose. A volume of culture (7 ml) wastransferred to 250 ml fresh 2× YT medium containing 34 μg/mlchloramphenicol and 0.1% glucose. After 3 hours of incubation, 0.5 mMIPTG was added to induce Fab expression. The culture was incubatedovernight at 25° C. Following incubation, the culture was centrifuged topellet the bacterial cells, and the pellet was resuspended in a BugBuster® lysis buffer (Novagen, Madison, Wis.). After centrifugation, thebacterial lysis supernatant was filtered, and the Fab fragments wereaffinity-purified through a Ni-NTA column (Qiagen, Valencia, Calif.)according to the manufacturer's instruction.

Other examples of protease-regulated antibodies were also constructedusing tandem linked variable regions from 3E10 and 1909. Theseantibodies contained, for example, V_(L)3E10-DDDDK-V_(L)19G9-CL on thelight chain and V_(H)3E10-DDDDK-V_(H)19G9-CH1-Myc-His6 on the heavychain, where the linker is cleavable by enterokinase, and Myc and His6are tags for detection and purification. An antibody library was alsoconstructed using the framework regions (FR), for example, FR4 of 3E10and FR1 of 19G9 either intact or truncated. Several types ofprotease-regulated antibodies were screened from this library. Thecloning, expression, and purification were performed as described above.

Example 2 Cloning and Expression of IgG-Like Antibodies

The expression vector pIE_SRgamma_fa contains cDNAs encoding theconstant regions of human IgG1 (fa haplotype) and kappa chains,respectively. An overlap PCR was performed to link the variable regionsof anti-TF antibody 3E10 and anti-RG1 antibody 19G9. The native signalpeptide of 19G9 was used for secretion of the protease-regulatedantibodies. Four examples of peptide linkers located between thevariable regions of 3E10 and 19G9 are Linker 1: SDDDDK (SEQ ID NO: 2),Linker 2: GGGGSDDDDK (SEQ ID NO: 3), Linker 3: GGGGSDDDDKGGGGS (SEQ IDNO: 4), and Linker 4: GGGGSGGGGSGGGGS (SEQ ID NO: 5). The primers foramplification of the variable region of the light chain introduced HindIII and Bsiw I sites into the 5′ and 3′ ends of PCR fragment,respectively. The resulting PCR-amplified V_(L) genes were cloned intothe HindIII/Bsiw site of pIE_SRgamma1_fa to createpIE-3E10V_(L)-linker-19G9V_(L). The same strategy was used to clone inframe V_(H) fusions of 3E10 and 19G9 (including linkers 1-4) intopIE-3E10V_(L)-linker-19G9V_(L). Briefly, the primer pairs of thevariable regions of 3E10 and 19G9 contained NotI/ApaI sites. The PCRproducts were digested with NotI/ApaI and inserted upstream of the CHregion of pIE-3E10V_(H)-linker-19G9V_(H) ensuring that the V_(H) regionswere in frame with the CH region in the respective pIE derivatives. Thefinal constructs were verified by DNA sequencing analysis.

Transfection and transient expression of the protease-regulatedantibodies were conducted using mammalian cells. Approximately 4×10⁸CHO-S cells supplemented with CHO-SF medium were prepared fortransfection. Transfection was carried out using Lipofectamine™ 2000(Invitrogen, Carlsbad, Calif.) and 1 mg plasmid DNA following themanufacturer's instruction. The cells were grown for three days aftertransfection, and the culture media was harvested and filtered forantibody isolation and purification.

Examples of the protease-regulated antibodies are described in Tables3-9

TABLE 3 PROTEASE-REGULATED ANTIBODIES (Type 1) Light chain Heavy chainFab-like protease-regulated antibodies against TF (3E10)DIVLTQPHSVSASPGKTVTISCTRSSGSVA DLVESGGTLVQPGGSLRLSCAASGFSFTDAWSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP MSWVRQAPGKELEWVSSISGSGGSTYYAGSVDRFSGSIDTSSNSASLTISGLKTEDEADYY KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCQSYDSNNLVVFGGGTKLTVLGAGGGGSDD CARVLSLTDYYWYGMDVWGQGTLVTVSASDDDDKRTVAAPSVFIFPPSDEQLKSGTASVVC DDKSSASTKGPSVFPLAPSSKSTSGGTAALGLLNNFYPREAKVQWKVDNALQSGNSQESVT CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLEQDSKDSTYSLSSTLTLSKADYEKHKVYAC QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKEVTHQGLSSPVTKSFNRGEC PSNTKVDKKVEPKCEF (SEQ ID NO: 33) (SEQ ID NO: 34)Fab-like protease-regulated antibodies against RG1 (19G9)DIVLTQSPGTLSLSPGERATLSCRASQSVSS QLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVSYLAWYQQKPGQAPRLLIYGASSRATGIPDR MHWLRQAPGKGLEWVSVIGTGGVTHYADSVKFSGSGSGTDFTLTISRLEPEDFAVYYCQQYS GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSSLTFGGGTKVEIKDDDDKRTVAAPSVFIFP ARWGYYGSGSYENDAFDIWGQGTMVTVDDDDPSDEQLKSGTASVVCLLNNFYPREAKVQWKV KSSASTKGPSVFPLAPSSKSTSGGTAALGCLDNALQSGNSQESVTEQDSKDSTYSLSSTLTL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSKADYEKHKVYACEVTHQGLSSPVTKSFNRG SGLYSLSSVVTVPSSSLGTQTYICNVNHKPS ECNTKVDKKVEPKCEF (SEQ ID NO: 35) (SEQ ID NO: 36)IgG-like protease-regulated antibodies against TF (3E10)NFMLTQPHSVSASPGKTVTISCTRSSGSVAS QVNLRESGGTLVQPGGSLRLSCAASGFSFTDYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDR AWMSWVRQAPGKELEWVSSISGSGGSTYYAGFSGSIDTSSNSASLTISGLKTEDEADYYCQS SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYDSNNLVVFGGGTKLTVLGQSDDDDKPKAAP YYCARVLSLTDYYWYGMDVWGQGTLVTVSASSVTLFPPSSEELQANKATLVCLISDFYPGAV DDDDKTKGPSVFPLAPSSKSTSGGTAALGCLTVAWKADSSPVKAGVETTTPSKQSNNKYAAS VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV SGLYSLSSVVTVPSSSLGTQTYICNVNHKPS APTECSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 37)SVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38)IgG-like protease-regulated antibodies against RG1 (19G9)EIVLTQSPGTLSLSPGERATLSCRASQSVS EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSSYLAWYQQKPGQAPRLLIYGASSRATGIP SYVMHWLRQAPGKGLEWVSVIGTGGVTHYADRFSGSGSGTDFTLTISRLEPEDFAVYYCQ DSVKGRFTISRDNAKNSLYLQMNSLRAEDTQYSSSLTFGGGTKVEIKRTSDDDDKVAAPS AVYYCARWGYYGSGSYENDAFDIWGQGTMVVFIFPPSDEQLKSGTASVVCLLNNFYPREA TVSSASDDDDDKTKGPSVFPLAPSSKSTSGKVQWKVDNALQSGNSQESVTEQDSKDSTYS GTAALGCLVKDYFPEPVTVSWNSGALTSGVLSSTLTLSKADYEKHKVYACEVTHQGLSSP HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT VTKSFNRGECYICNVNHKPSNTKVDKRVEPKSCDKTHTCP (SEQ ID NO: 39)PCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 40)

TABLE 4 PROTEASE-REGULATED ANTIBODIES (Type 2)Fab-like protease-regulated antibodies against TF and RG1 Light chainHeavy chain H1L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVAQVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKETAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSSVSASDDDDKEVQLVQSGGGLVQPGGSLRLS SYLAWYQQKPGQAPRLLIYGASSRATGIPDCAGSGFTFSSYVMHWLRQAPGKGLEWVSVI RFSGSGSGTDFTLTISRLEPEDFAVYYCQQGTGGVTHYADSVKGRFTISRDNAKNSLYLQ YSSSLTFGGGTKVEIKRTVAAPSVFIFPPSMNSLRAEDTAVYYCARWGYYGSGSYENDAF DEQLKSGTASVVCLLNNFYPREAKVQWKVDDIWGQGTMVTVSSASTKGPSVFPLAPSSKS NALQSGNSQESVTEQDSKDSTYSLSSTLTLTSGGTAALGCLVKDYFPEPVTVSWNSGALT SKADYEKHKVYACEVTHQGLSSPVTKSFNRSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG GEC TQTYICNVNHKPSNTKVDKKVEPKCEF(SEQ ID NO: 41) (SEQ ID NO: 42) H1L4 DIVLTQPHSVSASPGKTVTISCTRSSGSVAQVQLVESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKLTAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYLVSASDDDDKEVQLVQSGGGLVQPGGSLRLS AWYQQKPGQAPRLLIYGASSRATGIPDRFSCAGSGFTFSSYVMHWLRQAPGKGLEWVSVI GSGSGTDFTLTISRLEPEDFAVYYCQQYSSGTGGVTHYADSVKGRFTISRDNAKNSLYLQ SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQMNSLRAEDTAVYYCARWGYYGSGSYENDAF LKSGTASVVCLLNNFYPREAKVQWKVDNALDIWGQGTMVTVSSASTKGPSVFPLAPSSKS QSGNSQESVTEQDSKDSTYSLSSTLTLSKATSGGTAALGCLVKDYFPEPVTVSWNSGALT DYEKHKVYACEVTHQGLSSPVTKSFNRGECSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG (SEQ ID NO: 43)TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 44) H1L7DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVTPGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKEVQLVQSGGGLVQPGGSLRLSQQKPGQAPRLLIYGASSRATGIPDRFSGSG CAGSGFTFSSYVMHWLRQAPGKGLEWVSVISGTDFTLTISRLEPEDFAVYYCQQYSSSLT GTGGVTHYADSVKGRFTISRDNAKNSLYLQFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS MNSLRAEDTAVYYCARWGYYGSGSYENDAFGTASVVCLLNNFYPREAKVQWKVDNALQSG DIWGQGTMVTVSSASTKGPSVFPLAPSSKSNSQESVTEQDSKDSTYSLSSTLTLSKADYE TSGGTAALGCLVKDYFPEPVTVSWNSGALTKHKVYACEVTHQGLSSPVTKSFNRGEC SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG(SEQ ID NO: 45) TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 46) H4L2DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVTLTQSPGTLSLSPGERATLSCRASQSVSSSY VDDDDKQSGGGLVQPGGSLRLSCAGSGFTFLAWYQQKPGQAPRLLIYGASSRATGIPDRF SSYVMHWLRQAPGKGLEWVSVIGTGGVTHYSGSGSGTDFTLTISRLEPEDFAVYYCQQYS ADSVKGRFTISRDNAKNSLYLQMNSLRAEDSSLTFGGGTKVEIKRTVAAPSVFIFPPSDE TAVYYCARWGYYGSGSYENDAFDIWGQGTMQLKSGTASVVCLLNNFYPREAKVQWKVDNA VTVSSASTKGPSVFPLAPSSKSTSGGTAALLQSGNSQESVTEQDSKDSTYSLSSTLTLSK GCLVKDYFPEPVTVSWNSGALTSGVHTFPAADYEKHKVYACEVTHQGLSSPVTKSFNRGE VLQSSGLYSLSSVVTVPSSSLGTQTYICNV CNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 47) (SEQ ID NO: 48) H4L5DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVTSPGTLSLSPGERATLSCRASQSVSSSYLAW VDDDDKQSGGGLVQPGGSLRLSCAGSGFTFYQQKPGQAPRLLIYGASSRATGIPDRFSGS SSYVMHWLRQAPGKGLEWVSVIGTGGVTHYGSGTDFTLTISRLEPEDFAVYYCQQYSSSL ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK TAVYYCARWGYYGSGSYENDAFDIWGQGTMSGTASVVCLLNNFYPREAKVQWKVDNALQS VTVSSASTKGPSVFPLAPSSKSTSGGTAALGNSQESVTEQDSKDSTYSLSSTLTLSKADY GCLVKDYFPEPVTVSWNSGALTSGVHTFPAEKHKVYACEVTHQGLSSPVTKSFNRGEC VLQSSGLYSLSSVVTVPSSSLGTQTYICNV(SEQ ID NO: 49) NHKPSNTKVDKKVEPKCEF (SEQ ID NO: 50) H4L7DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVTPGTLSLSPGERATLSCRASQSVSSSYLAWY VDDDDKQSGGGLVQPGGSLRLSCAGSGFTFQQKPGQAPRLLIYGASSRATGIPDRFSGSG SSYVMHWLRQAPGKGLEWVSVIGTGGVTHYSGTDFTLTISRLEPEDFAVYYCQQYSSSLT ADSVKGRFTISRDNAKNSLYLQMNSLRAEDFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS TAVYYCARWGYYGSGSYENDAFDIWGQGTMGTASVVCLLNNFYPREAKVQWKVDNALQSG VTVSSASTKGPSVFPLAPSSKSTSGGTAALNSQESVTEQDSKDSTYSLSSTLTLSKADYE GCLVKDYFPEPVTVSWNSGALTSGVHTFPAKHKVYACEVTHQGLSSPVTKSFNRGEC VLQSSGLYSLSSVVTVPSSSLGTQTYICNV(SEQ ID NO: 51) NHKPSNTKVDKKVEPKCEF (SEQ ID NO: 52) H5L5DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVTSPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKLVQPGGSLRLSCAGSGFTFSSYQQKPGQAPRLLIYGASSRATGIPDRFSGS YVMHWLRQAPGKGLEWVSVIGTGGVTHYADGSGTDFTLTISRLEPEDFAVYYCQQYSSSL SVKGRFTISRDNAKNSLYLQMNSLRAEDTATFGGGTKVEIKRTVAAPSVFIFPPSDEQLK VYYCARWGYYGSGSYENDAFDIWGQGTMVTSGTASVVCLLNNFYPREAKVQWKVDNALQS VSSASTKGPSVFPLAPSSKSTSGGTAALGCGNSQESVTEQDSKDSTYSLSSTLTLSKADY LVKDYFPEPVTVSWNSGALTSGVHTFPAVLEKHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH(SEQ ID NO: 53) KPSNTKVDKKVEPKCEF (SEQ ID NO: 54)

TABLE 5 PROTEASE-REGULATED ANTIBODIES (Type 2)IgG-like protease-regulated antibodies against TF and RG1 Light chainHeavy chain 3E10-Linkerl-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVAQVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKETAVYYCARVLSLTDYYWYGMDVWGQGTLVT IVLTQSPGTLSLSPGERATLSCRASQSVSSVSASDDDDKEVQLVQSGGGLVQPGGSLRLS SYLAWYQQKPGQAPRLLIYGASSRATGIPDCAGSGFTFSSYVMHWLRQAPGKGLEWVSVI RFSGSGSGTDFTLTISRLEPEDFAVYYCQQGTGGVTHYADSVKGRFTISRDNAKNSLYLQ YSSSLTFGGGTKVEIKRTVAAPSVFIFPPSMNSLRAEDTAVYYCARWGYYGSGSYENDAF DEQLKSGTASVVCLLNNFYPREAKVQWKVDDIWGQGTMVTVSSASTKGPSVFPLAPSSKS NALQSGNSQESVTEQDSKDSTYSLSSTLTLTSGGTAALGCLVKDYFPEPVTVSWNSGALT SKADYEKHKVYACEVTHQGLSSPVTKSFNRSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG GEC TQTYICNVNHKPSNTKVDKRVEPKSCDKTH(SEQ ID NO: 55) TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 56) 3E10-Linker2-19G9NFMLTQPHSVSASPGKTVTISCTRSSGSVA QVNLRESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGAGGGGSDD TAVYYCARVLSLTDYYWYGMDVWGQGTLVTDDKEIVLTQSPGTLSLSPGERATLSCRASQ VSAGGGGSDDDDKEVQLVQSGGGLVQPGGSSVSSSYLAWYQQKPGQAPRLLIYGASSRAT LRLSCAGSGFTFSSYVMHWLRQAPGKGLEWGIPDRFSGSGSGTDFTLTISRLEPEDFAVY VSVIGTGGVTHYADSVKGRFTISRDNAKNSYCQQYSSSLTFGGGTKVEIKRTVAAPSVFI LYLQMNSLRAEDTAVYYCARWGYYGSGSYEFPPSDEQLKSGTASVVCLLNNFYPREAKVQ NDAFDIWGQGTMVTVSSASTKGPSVFPLAPWKVDNALQSGNSQESVTEQDSKDSTYSLSS SSKSTSGGTAALGCLVKDYFPEPVTVSWNSTLTLSKADYEKHKVYACEVTHQGLSSPVTK GALTSGVHTFPAVLQSSGLYSLSSVVTVPS SFNRGECSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 57)DKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 58)3E10-Linker3-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVAQVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGAGGGGSDDTAVYYCARVLSLTDYYWYGMDVWGQGTLVT DDKGGGGSEIVLTQSPGTLSLSPGERATLSVSAGGGGSDDDDKGGGGSEVQLVQSGGGLV CRASQSVSSSYLAWYQQKPGQAPRLLIYGAQPGGSLRLSCAGSGFTFSSYVMHWLRQAPG SSRATGIPDRFSGSGSGTDFTLTISRLEPEKGLEWVSVIGTGGVTHYADSVKGRFTISRD DFAVYYCQQYSSSLTFGGGTKVEIKRTVAANAKNSLYLQMNSLRAEDTAVYYCARWGYYG PSVFIFPPSDEQLKSGTASVVCLLNNFYPRSGSYENDAFDIWGQGTMVTVSSASTKGPSV EAKVQWKVDNALQSGNSQESVTEQDSKDSTFPLAPSSKSTSGGTAALGCLVKDYFPEPVT YSLSSTLTLSKADYEKHKVYACEVTHQGLSVSWNSGALTSGVHTFPAVLQSSGLYSLSSV SPVTKSFNRGECVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 59)EPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60)3E10-Linker4-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVASYYVQQVNLRESGGTLVQPGGSLRLSCAASGFSFTDAWMS WYQQRPGSSPTTVIYEDNHRPSGVPDRFSGSIDTSWVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTI SNSASLTISGLKTEDEADYYCQSYDSNNLVVFGGGSRDNSKNTLYLQMNSLRAEDTAVYYCARVLSLTDY TKLTVLGAGGGGSGGGGSGGGGSEIVLTQSPGTLSYWYGMDVWGQGTLVTVSAGGGGSGGGGSGGGGSEV LSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPERQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRD DFAVYYCQQYSSSLTFGGGTKVEIKRTVAAPSVFINAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYE FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNNDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKST ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYESGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF KHKVYACEVTHQGLSSPVTKSFNRGECPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK (SEQ ID NO: 61)PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62) 3E10-Link1-19G9 FabDIVLTQPHSVSASPGKTVTISCTRSSGSVASYYVQ QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMSWYQQRPGSSPTTVIYEDNHRPSGVPDRFSGSIDTS WVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTISNSASLTISGLKTEDEADYYCQSYDSNNLVVFGGG SRDNSKNTLYLQMNSLRAEDTAVYYCARVLSLTDYTKLTVLGASDDDDKEIVLTQSPGTLSLSPGERATL YWYGMDVWGQGTLVTVSASDDDDKEVQLVQSGGGLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRA VQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLETGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ WVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQYSSSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK MNSLRAEDTAVYYCARWGYYGSGSYENDAFDIWGQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCSVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLTHQGLSSPVTKSFNRGEA YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK (SEQ ID NO: 63)VEPKSEF (SEQ ID NO: 64)

TABLE 6 PROTEASE-REGULATED ANTIBODIES (Type 3)Fab-like protease-regulated antibodies Light chain Heavy chain H1L5DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVTSPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKEVQLVQSGGGLVQPGGSLRLSYQQKPGQAPRLLIYGASSRATGIPDRFSGS CAGSGFTFSSYVMHWLRQAPGKGLEWVSVIGSGTDFTLTISRLEPEDFAVYYCQQYSSSL GTGGVTHYADSVKGRFTISRDNAKNSLYLQTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK MNSLRAEDTAVYYCARWGYYGSGSYENDAFSGTASVVCLLNNFYPREAKVQWKVDNALQS DIWGQGTMVTVSSASTKGPSVFPLAPSSKSGNSQESVTEQDSKDSTYSLSSTLTLSKADY TSGGTAALGCLVKDYFPEPVTVSWNSGALTEKHKVYACEVTHQGLSSPVTKSFNRGEC SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG(SEQ ID NO: 65) TQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 66) H2L1DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVTIVLTQSPGTLSLSPGERATLSCRASQSVSS VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGSYLAWYQQKPGQAPRLLIYGASSRATGIPD SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GVTHYADSVKGRFTISRDNAKNSLYLQMNSYSSSLTFGGGTKVEIKRTVAAPSVFIFPPS LRAEDTAVYYCARWGYYGSGSYENDAFDIWDEQLKSGTASVVCLLNNFYPREAKVQWKVD GQGTMVTVSSASTKGPSVFPLAPSSKSTSGNALQSGNSQESVTEQDSKDSTYSLSSTLTL GTAALGCLVKDYFPEPVTVSWNSGALTSGVSKADYEKHKVYACEVTHQGLSSPVTKSFNR HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT GECYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 67) (SEQ ID NO: 68) H2L2DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVTLTQSPGTLSLSPGERATLSCRASQSVSSSY VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGLAWYQQKPGQAPRLLIYGASSRATGIPDRF SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGSGSGSGTDFTLTISRLEPEDFAVYYCQQYS GVTHYADSVKGRFTISRDNAKNSLYLQMNSSSLTFGGGTKVEIKRTVAAPSVFIFPPSDE LRAEDTAVYYCARWGYYGSGSYENDAFDIWQLKSGTASVVCLLNNFYPREAKVQWKVDNA GQGTMVTVSSASTKGPSVFPLAPSSKSTSGLQSGNSQESVTEQDSKDSTYSLSSTLTLSK GTAALGCLVKDYFPEPVTVSWNSGALTSGVADYEKHKVYACEVTHQGLSSPVTKSFNRGE HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT CYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 69) (SEQ ID NO: 70) H2L4DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASASDDDD TAVYYCARVLSLTDYYWYGMDVWGQGTLVTKLTQSPGTLSLSPGERATLSCRASQSVSSS VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGYLAWYQQKPGQAPRLLIYGASSRATGIPDR SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GVTHYADSVKGRFTISRDNAKNSLYLQMNSSSSLTFGGGTKVEIKRTVAAPSVFIFPPSD LRAEDTAVYYCARWGYYGSGSYENDAFDIWEQLKSGTASVVCLLNNFYPREAKVQWKVDN GQGTMVTVSSASTKGPSVFPLAPSSKSTSGALQSGNSQESVTEQDSKDSTYSLSSTLTLS GTAALGCLVKDYFPEPVTVSWNSGALTSGVKADYEKHKVYACEVTHQGLSSPVTKSFNRG HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT ECYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 71) (SEQ ID NO: 72) H2L5DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVTSPGTLSLSPGERATLSCRASQSVSSSYLAW VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGYQQKPGQAPRLLIYGASSRATGIPDRFSGS SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGGSGTDFTLTISRLEPEDFAVYYCQQYSSSL GVTHYADSVKGRFTISRDNAKNSLYLQMNSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK LRAEDTAVYYCARWGYYGSGSYENDAFDIWSGTASVVCLLNNFYPREAKVQWKVDNALQS GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGNSQESVTEQDSKDSTYSLSSTLTLSKADY GTAALGCLVKDYFPEPVTVSWNSGALTSGVEKHKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT(SEQ ID NO: 73) YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 74) H2L7DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVTPGTLSLSPGERATLSCRASQSVSSSYLAWY VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGQQKPGQAPRLLIYGASSRATGIPDRFSGSG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGSGTDFTLTISRLEPEDFAVYYCQQYSSSLT GVTHYADSVKGRFTISRDNAKNSLYLQMNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS LRAEDTAVYYCARWGYYGSGSYENDAFDIWGTASVVCLLNNFYPREAKVQWKVDNALQSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE GTAALGCLVKDYFPEPVTVSWNSGALTSGVKHKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT(SEQ ID NO: 75) YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 76) H2L8DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKSPG TAVYYCARVLSLTDYYWYGMDVWGQGTLVTTLSLSPGERATLSCRASQSVSSSYLAWYQQ VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGKPGQAPRLLIYGASSRATGIPDRFSGSGSG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGTDFTLTISRLEPEDFAVYYCQQYSSSLTFG GVTHYADSVKGRFTISRDNAKNSLYLQMNSGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT LRAEDTAVYYCARWGYYGSGSYENDAFDIWASVVCLLNNFYPREAKVQWKVDNALQSGNS GQGTMVTVSSASTKGPSVFPLAPSSKSTSGQESVTEQDSKDSTYSLSSTLTLSKADYEKH GTAALGCLVKDYFPEPVTVSWNSGALTSGVKVYACEVTHQGLSSPVTKSFNRGEC HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 77)YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 78)

TABLE 7 PROTEASE-REGULATED ANTIBODIES (Type 3)IgG-like protease-regulated antibodies Light chain Heavy chain3E10-Linkerl a-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVAQVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVSDDDDKEIVLTAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYLVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG AWYQQKPGQAPRLLIYGASSRATGIPDRFSSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GSGSGTDFTLTISRLEPEDFAVYYCQQYSSGVTHYADSVKGRFTISRDNAKNSLYLQMNS SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLRAEDTAVYYCARWGYYGSGSYENDAFDIW LKSGTASVVCLLNNFYPREAKVQWKVDNALGQGTMVTVSSASTKGPSVFPLAPSSKSTSG QSGNSQESVTEQDSKDSTYSLSSTLTLSKAGTAALGCLVKDYFPEPVTVSWNSGALTSGV DYEKHKVYACEVTHQGLSSPVTKSFNRGECHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT (SEQ ID NO: 79)YICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK(SEQ ID NO: 80) 3E10-Linkerlb-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVAQVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVSDDDDKLTQSTAVYYCARVLSLTDYYWYGMDVWGQGTLVT PGTLSLSPGERATLSCRASQSVSSSYLAWYVDDDDKQSGGGLVQPGGSLRLSCAGSGFTF QQKPGQAPRLLIYGASSRATGIPDRFSGSGSSYVMHWLRQAPGKGLEWVSVIGTGGVTHY SGTDFTLTISRLEPEDFAVYYCQQYSSSLTADSVKGRFTISRDNAKNSLYLQMNSLRAED FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSTAVYYCARWGYYGSGSYENDAFDIWGQGTM GTASVVCLLNNFYPREAKVQWKVDNALQSGVTVSSASTKGPSVFFLAPSSKSTSGGTAAL NSQESVTEQDSKDSTYSLSSTLTLSKADYEGCLVKDYFPEPVTVSWNSGALTSGVHTFPA KHKVYACEVTHQGLSSPVTKSFNRGECVLQSSGLYSLSSVVTVPSSSLGTQTYICNV (SEQ ID NO: 81)NHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK(SEQ ID NO: 82) 3E10-Linkerlc-19G9 NFMLTQPHSVSASPGKTVTISCTRSSGSVAQVNLRESGGTLVQPGGSLRLSCAASGFSFT SYYVQWYQQRPGSSPTTVIYEDNHRPSGVPDAWMSWVRQAPGKELEWVSSISGSGGSTYY DRFSGSIDTSSNSASLTISGLKTEDEADYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED CQSYDSNNLVVFGGGTKLTVLGASDDDDKLTAVYYCARVLSLTDYYWYGMDVWGQGTLVT TQSPGTLSLSPGERATLSCRASQSVSSSYLVSASDDDDKQSGGGLVQPGGSLRLSCAGSG AWYQQKPGQAPRLLIYGASSRATGIPDRFSFTFSSYVMHWLRQAPGKGLEWVSVIGTGGV GSGSGTDFTLTISRLEPEDFAVYYCQQYSSTHYADSVKGRFTISRDNAKNSLYLQMNSLR SLTFGGGTKVEIKRTVAAPSVFIFPPSDEQAEDTAVYYCARWGYYGSGSYENDAFDIWGQ LKSGTASVVCLLNNFYPREAKVQWKVDNALGTMVTVSSASTKGPSVFPLAPSSKSTSGGT QSGNSQESVTEQDSKDSTYSLSSTLTLSKAAALGCLVKDYFPEPVTVSWNSGALTSGVHT DYEKHKVYACEVTHQGLSSPVTKSFNRGECFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (SEQ ID NO: 83)CNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK(SEQ ID NO: 84)

TABLE 8 PROTEASE-REGULATED ANTIBODIES (Type 4) Light chain Heavy chainH3L1 DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVTIVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKQSGGGLVQPGGSLRLSCAGSGSYLAWYQQKPGQAPRLLIYGASSRATGIPD FTFSSYVMHWLRQAPGKGLEWVSVIGTGGVRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ THYADSVKGRFTISRDNAKNSLYLQMNSLRYSSSLTFGGGTKVEIKRTVAAPSVFIFPPS AEDTAVYYCARWGYYGSGSYENDAFDIWGQDEQLKSGTASVVCLLNNFYPREAKVQWKVD GTMVTVSSASTKGPSVFPLAPSSKSTSGGTNALQSGNSQESVTEQDSKDSTYSLSSTLTL AALGCLVKDYFPEPVTVSWNSGALTSGVHTSKADYEKHKVYACEVTHQGLSSPVTKSFNR FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI GECCNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 85) (SEQ ID NO: 86) H3L2DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVTLTQSPGTLSLSPGERATLSCRASQSVSSSY VSASDDDDKQSGGGLVQPGGSLRLSCAGSGLAWYQQKPGQAPRLLIYGASSRATGIPDRF FTFSSYVMHWLRQAPGKGLEWVSVIGTGGVSGSGSGTDFTLTISRLEPEDFAVYYCQQYS THYADSVKGRFTISRDNAKNSLYLQMNSLRSSLTFGGGTKVEIKRTVAAPSVFIFPPSDE AEDTAVYYCARWGYYGSGSYENDAFDIWGQQLKSGTASVVCLLNNFYPREAKVQWKVDNA GTMVTVSSASTKGPSVFPLAPSSKSTSGGTLQSGNSQESVTEQDSKDSTYSLSSTLTLSK AALGCLVKDYFPEPVTVSWNSGALTSGVHTADYEKHKVYACEVTHQGLSSPVTKSFNRGE FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CCNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 87) (SEQ ID NO: 88) H3L4DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVTTQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKQSGGGLVQPGGSLRLSCAGSGAWYQQKPGQAPRLLIYGASSRATGIPDRFS FTFSSYVMHWLRQAPGKGLEWVSVIGTGGVGSGSGTDFTLTISRLEPEDFAVYYCQQYSS THYADSVKGRFTISRDNAKNSLYLQMNSLRSLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ AEDTAVYYCARWGYYGSGSYENDAFDIWGQLKSGTASVVCLLNNFYPREAKVQWKVDNAL GTMVTVSSASTKGPSVFPLAPSSKSTSGGTQSGNSQESVTEQDSKDSTYSLSSTLTLSKA AALGCLVKDYFPEPVTVSWNSGALTSGVHTDYEKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI(SEQ ID NO: 89) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 90) H3L5DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKLTQ TAVYYCARVLSLTDYYWYGMDVWGQGTLVTSPGTLSLSPGERATLSCRASQSVSSSYLAW VSASDDDDKQSGGGLVQPGGSLRLSCAGSGYQQKPGQAPRLLIYGASSRATGIPDRFSGS FTFSSYVMHWLRQAPGKGLEWVSVIGTGGVGSGTDFTLTISRLEPEDFAVYYCQQYSSSL THYADSVKGRFTISRDNAKNSLYLQMNSLRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK AEDTAVYYCARWGYYGSGSYENDAFDIWGQSGTASVVCLLNNFYPREAKVQWKVDNALQS GTMVTVSSASTKGPSVFPLAPSSKSTSGGTGNSQESVTEQDSKDSTYSLSSTLTLSKADY AALGCLVKDYFPEPVTVSWNSGALTSGVHTEKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI(SEQ ID NO: 91) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 92) H3L7DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVTPGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKQSGGGLVQPGGSLRLSCAGSGQQKPGQAPRLLIYGASSRATGIPDRFSGSG FTFSSYVMHWLRQAPGKGLEWVSVIGTGGVSGTDFTLTISRLEPEDFAVYYCQQYSSSLT THYADSVKGRFTISRDNAKNSLYLQMNSLRFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS AEDTAVYYCARWGYYGSGSYENDAFDIWGQGTASVVCLLNNFYPREAKVQWKVDNALQSG GTMVTVSSASTKGPSVFPLAPSSKSTSGGTNSQESVTEQDSKDSTYSLSSTLTLSKADYE AALGCLVKDYFPEPVTVSWNSGALTSGVHTKHKVYACEVTHQGLSSPVTKSFNRGEC FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI(SEQ ID NO: 93) CNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 94) H1L2DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKEIV TAVYYCARVLSLTDYYWYGMDVWGQGTLVTLTQSPGTLSLSPGERATLSCRASQSVSSSY VSASDDDDKEVQLVQSGGGLVQPGGSLRLSLAWYQQKPGQAPRLLIYGASSRATGIPDRF CAGSGFTFSSYVMHWLRQAPGKGLEWVSVISGSGSGTDFTLTISRLEPEDFAVYYCQQYS GTGGVTHYADSVKGRFTISRDNAKNSLYLQSSLTFGGGTKVEIKRTVAAPSVFIFPPSDE MNSLRAEDTAVYYCARWGYYGSGSYENDAFQLKSGTASVVCLLNNFYPREAKVQWKVDNA DIWGQGTMVTVSSASTKGPSVFPLAPSSKSLQSGNSQESVTEQDSKDSTYSLSSTLTLSK TSGGTAALGCLVKDYFPEPVTVSWNSGALTADYEKHKVYACEVTHQGLSSPVTKSFNRGE SGVHTFPAVLQSSGLYSLSSVVTVPSSSLG CTQTYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 95) (SEQ ID NO: 96) H5L1DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVTIVLTQSPGTLSLSPGERATLSCRASQSVSS VSASDDDDKLVQPGGSLRLSCAGSGFTFSSSYLAWYQQKPGQAPRLLIYGASSRATGIPD YVMHWLRQAPGKGLEWVSVIGTGGVTHYADRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ SVKGRFTISRDNAKNSLYLQMNSLRAEDTAYSSSLTFGGGTKVEIKRTVAAPSVFIFPPS VYYCARWGYYGSGSYENDAFDIWGQGTMVTDEQLKSGTASVVCLLNNFYPREAKVQWKVD VSSASTKGPSVFPLAPSSKSTSGGTAALGCNALQSGNSQESVTEQDSKDSTYSLSSTLTL LVKDYFPEPVTVSWNSGALTSGVHTFPAVLSKADYEKHKVYACEVTHQGLSSPVTKSFNR QSSGLYSLSSVVTVPSSSLGTQTYICNVNH GECKPSNTKVDKKVEPKCEF (SEQ ID NO: 97) (SEQ ID NO: 98) H5L4DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKL TAVYYCARVLSLTDYYWYGMDVWGQGTLVTTQSPGTLSLSPGERATLSCRASQSVSSSYL VSASDDDDKLVQPGGSLRLSCAGSGFTFSSAWYQQKPGQAPRLLIYGASSRATGIPDRFS YVMHWLRQAPGKGLEWVSVIGTGGVTHYADGSGSGTDFTLTISRLEPEDFAVYYCQQYSS SVKGRFTISRDNAKNSLYLQMNSLRAEDTASLTEGGGTKVEIKRTVAAPSVFIFPPSDEQ VYYCARWGYYGSGSYENDAFDIWGQGTMVTLKSGTASVVCLLNNFYPREAKVQWKVDNAL VSSASTKGPSVFPLAPSSKSTSGGTAALGCQSGNSQESVTEQDSKDSTYSLSSTLTLSKA LVKDYFPEPVTVSWNSGALTSGVHTFPAVLDYEKHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH(SEQ ID NO: 99) KPSNTKVDKKVEPKCEF (SEQ ID NO: 100) H5L7DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKS TAVYYCARVLSLTDYYWYGMDVWGQGTLVTPGTLSLSPGERATLSCRASQSVSSSYLAWY VSASDDDDKLVQPGGSLRLSCAGSGFTFSSQQKPGQAPRLLIYGASSRATGIPDRFSGSG YVMHWLRQAPGKGLEWVSVIGTGGVTHYADSGTDFTLTISRLEPEDFAVYYCQQYSSSLT SVKGRFTISRDNAKNSLYLQMNSLRAEDTAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS VYYCARWGYYGSGSYENDAFDIWGQGTMVTGTASVVCLLNNFYPREAKVQWKVDNALQSG VSSASTKGPSVFPLAPSSKSTSGGTAALGCNSQESVTEQDSKDSTYSLSSTLTLSKADYE LVKDYFPEPVTVSWNSGALTSGVHTFPAVLKHKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH(SEQ ID NO: 101) KPSNTKVDKKVEPKCEF (SEQ ID NO: 102) H5L8DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGDDDDKSPG TAVYYCARVLSLTDYYWYGMDVWGQGTLVTTLSLSPGERATLSCRASQSVSSSYLAWYQQ VSASDDDDKLVQPGGSLRLSCAGSGFTFSSKPGQAPRLLIYGASSRATGIPDRFSGSGSG YVMHWLRQAPGKGLEWVSVIGTGGVTHYADTDFTLTISRLEPEDFAVYYCQQYSSSLTFG SVKGRFTISRDNAKNSLYLQMNSLRAEDTAGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT VYYCARWGYYGSGSYENDAFDIWGQGTMVTASVVCLLNNFYPREAKVQWKVDNALQSGNS VSSASTKGPSVFPLAPSSKSTSGGTAALGCQESVTEQDSKDSTYSLSSTLTLSKADYEKH LVKDYFPEPVTVSWNSGALTSGVHTFPAVLKVYACEVTHQGLSSPVTKSFNRGEC QSSGLYSLSSVVTVPSSSLGTQTYICNVNH(SEQ ID NO: 103) KPSNTKVDKKVEPKCEF (SEQ ID NO: 104) H6L1DIVLTQPHSVSASPGKTVTISCTRSSGSVA QVQLVESGGTLVQPGGSLRLSCAASGFSFTSYYVQWYQQRPGSSPTTVIYEDNHRPSGVP DAWMSWVRQAPGKELEWVSSISGSGGSTYYDRFSGSIDTSSNSASLTISGLKTEDEADYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDCQSYDSNNLVVFGGGTKLTVLGASDDDDKE TAVYYCARVLSLTDYYWYGMDVWGQGTLVTIVLTQSPGTLSLSPGERATLSCRASQSVSS VDDDDKLVQPGGSLRLSCAGSGFTFSSYVMSYLAWYQQKPGQAPRLLIYGASSRATGIPD HWLRQAPGKGLEWVSVIGTGGVTHYADSVKRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ GRFTISRDNAKNSLYLQNNSLRAEDTAVYYYSSSLTFGGGTKVEIKRTVAAPSVFIFPPS CARWGYYGSGSYENDAFDIWGQGTMVTVSSDEQLKSGTASVVCLLNNFYPREAKVQWKVD ASTKGPSVFPLAPSSKSTSGGTAALGCLVKNALQSGNSQESVTEQDSKDSTYSLSSTLTL DYFPEPVTVSWNSGALTSGVHTFPAVLQSSSKADYEKHKVYACEVTHQGLSSPVTKSFNR GLYSLSSVVTVPSSSLGTQTYICNVNHKPS GECNTKVDKKVEPKCEF (SEQ ID NO: 105) (SEQ ID NO: 106)

TABLE 9 PROTEASE-REGULATED ANTIBODIES (Type 4) Heavy chain Heavy chainH1L5a QVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVTVSASDDDDKEVQLVQSGGGLVQPGGSLRLS VSASDDDDKEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLEWVSVI CAGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQ GTGGVTHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYENDAF MNSLRAEDTAVYYCARWGYYGSGSYENDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS DIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKCEF TQTYICNVMHKPSNTICVDKICVEPKCEF(SEQ ID NO: 107) (SEQ ID NO: 108) H2L1a QVQLVESGGTLVQPGGSLRLSCAASGFSFTQVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYYDAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDAGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVTTAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNSGVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIWLRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEFYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 109) (SEQ ID NO: 110) H2L2aQVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVTVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 111)(SEQ ID NO: 112) H2L4a QVQLVESGGTLVQPGGSLRLSCAASGFSFTQVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYYDAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDAGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVTTAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNSGVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIWLRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEFYICNVNHKPSNTICVDKICVEPKCEF (SEQ ID NO: 113) (SEQ ID NO: 114) H2L5aQVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVTVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 115)(SEQ ID NO: 116) H2L7a QVQLVESGGTLVQPGGSLRLSCAASGFSFTQVQLVESGGTLVQPGGSLRLSCAASGFSFT DAWMSWVRQAPGKELEWVSSISGSGGSTYYDAWMSWVRQAPGKELEWVSSISGSGGSTYY AGSVKGRFTISRDNSKNTLYLQMNSLRAEDAGSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARAAAAAAAAAAAAAAWGQGTLVTTAVYYCARAAAAAAAAAAAAAAWGQGTLVT VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG GVTHYADSVKGRFTISRDNAKNSLYLQMNSGVTHYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARWGYYGSGSYENDAFDIWLRAEDTAVYYCARWGYYGSGSYENDAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKCEFYICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 117) (SEQ ID NO: 118) H2L8aQVQLVESGGTLVQPGGSLRLSCAASGFSFT QVQLVESGGTLVQPGGSLRLSCAASGFSFTDAWMSWVRQAPGKELEWVSSISGSGGSTYY DAWMSWVRQAPGKELEWVSSISGSGGSTYYAGSVKGRFTISRDNSKNTLYLQMNSLRAED AGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAAAAAAAAAAAAAAWGQGTLVT TAVYYCARAAAAAAAAAAAAAAWGQGTLVTVDDDDKEVQLVQSGGGLVQPGGSLRLSCAG VDDDDKEVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYVMHWLRQAPGKGLEWVSVIGTG SGFTFSSYVMHWLRQAPGKGLEWVSVIGTGGVTHYADSVKGRFTISRDNAKNSLYLQMNS GVTHYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARWGYYGSGSYENDAFDIW LRAEDTAVYYCARWGYYGSGSYENDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKCEF YICNVNHKPSNTKVDKKVEPKCEF (SEQ ID NO: 119)(SEQ ID NO: 120)

Example 3 TF-Binding ELISA

Biotinylated TF (1 μg/ml) was added to streptavidin pre-coated 96-wellplates (Pierce Chemical, Rockford, Ill.) and incubated for 1 hr. Theplates were then washed (5×) with PBS containing 0.5% Tween-20. Samplesand controls (serially diluted) were added to the wells and incubatedfor 1 hr, followed by washes (5×) with PBS containing 0.5% Tween-20.Horseradish peroxidase (HRP)-conjugated anti-human IgG or HRP-conjugatedanti-human Fab were diluted in PBS (1:5000) and added to each well.Following a 1 hr incubation, the plates were washed again. Amplex Red(10 μg/ml) was added to each well, and the signal was read using a platereader. The data was analyzed using Softmax (Molecular Devices,Sunnyvale, Calif.). Results are shown in FIG. 10.

Example 4 RG1-Binding ELISA

Ninety-six well plates were coated with RG1 (1 μg/ml) by overnightincubation, and the plates were then washed (5×) with PBS containing0.5% Tween-20. Samples and controls (serially diluted) were added to thewells and incubated for 1 hr, followed by washes (5×) with PBScontaining 0.5% Tween-20. Horserandish peroxidase (HRP)-conjugatedanti-human IgG or HRP-conjugated anti-human Fab were diluted in PBS(1:5000) and added to each well. Following a 1 hr incubation, the plateswere washed again. Amplex Red (10 μg/ml) was added to each well, and thesignal was read using a plate reader. The data was analyzed usingSoftmax (Molecular Devices, Sunnyvale, Calif.). Results are shown inFIG. 11.

Example 5 Sandwich Antigen-Binding ELISA

The antigen binding activity of a bispecific protease-regulated antibody(illustrated in FIG. 2) was measured using a sandwich antigen-bindingELISA. This antibody binds two antigens, RG-1 and TF, and the linkercontains cleavage sites for enterokinase (“EK”).

Ninety-six well plates were coated with RG1 (1 μg/ml) by overnightincubation. The plates were then washed five times with PBS containing0.5% Tween-20. Antibody samples and controls were digested with 30 unitsof enterokinase for 16 hr at 37° C. (see Example 4). The antibodysamples, with or without enterokinase digestion, were serially dilutedand added to the wells of the ELISA plates. The samples were incubatedfor one hour, followed by washes (5×) with PBS containing 0.5% Tween-20.Biotinylated TF (0.1 μg/ml) was added to each well and incubated for onehour. Horseradish peroxidase (HRP)-conjugated streptavidin (1:10000diluted) was then added to each well. Following a one-hour incubation,the plates were washed again. Amplex Red (10 μg/ml) was added to eachwell, and the signal was read using a plate reader. The data wasanalyzed using Softmax® (Molecular Devices, Sunnyvale, Calif.). Parentalantibodies 3E10, 19G9, and polyclonal human Fab were used as controls.The results are the average of duplicate wells (FIG. 12). The untreatedbispecific protease-regulated antibody simultaneously binds to both TFand RG-1 (“Link 1” and “Link2,” respectively). However, followingenterokinase treatment, the binding to both antigens is greatly reduced(“Link1/EK” and “Link2/EK,” respectively).

The antigen binding activity of several examples of protease-regulatedantibodies was also measured using this assay. For example, the antigenbinding activity of protease-regulated antibodies 3E10-Type1-Fab and19G9-Type1-Fab is shown in FIG. 13. The controls are designated3E10-Reg-Fab, 19G9-Reg-Fab, and HuFab.

The antigen binding activity of Fab-like protease-regulated antibodiesis demonstrated in FIG. 14. The activity of antibodies H1L1, H1L4, H1L7,H4L7, and H5L5 (Type 2) was measured in the absence and presence ofenterokinase. Parental antibodies 3E10, 19G9, and polyclonal human Fabwere used as controls. Similarly, FIG. 15 shows the antigen bindingactivity of Fab-like protease-regulated antibodies H2L1, H2L2, and H2L8(Type 3) and H3L1, H3L4, and H5L4 (Type 4).

Example 6 Enterokinase Digestion of Protease-Regulated Antibodies

Protease-regulated antibodies were digested with EnterokinaseMax™, thecatalytic subunit of enterokinase (Invitrogen, Carlsbad, Calif.). Theconcentration of antibodies was adjusted to 1-5 μg/ml. A volume ofantibody (100 μl) was mixed with 20 μl 10× EnterokinaseMax™ buffer and75 μl sterile water in a tube. EnterokinaseMax™ (5 μl) was added to eachsample and the samples were incubated at 37° C. for 16 hr. For thecontrol group, a volume of water (5 μl) was used.

Example 7 Western Blots of Antibodies

Three detection antibodies were used to probe protease-regulatedantibodies: anti-human kappa antibody, anti-human IgG(H+L), and anti-Myctag antibodies. These detection antibodies were conjugated withhorseradish peroxidase (HRP). Approximately 50 ng of antibody sampleswere mixed with loading buffer containing DTT (Invitrogen, Carlsbad,Calif.) and boiled for 5 min. The samples were then loaded onto a 12%Bis-Tris NuPAGE® gel (Invitrogen, Carlsbad, Calif.), separated, andtransferred to nitrocellulose membranes. After blocking with 5% dry milkfor 2 hr, the nitrocellulose membrane was incubated with a detectionantibody for 1.5 hr. The membrane was then washed in PBS containing 0.5%Tween-20, and incubated with SuperSignal West Femto (Pierce Chemical,Rockford, Ill.), and expose to X-ray film for development. Results areshown in FIGS. 16-18.

Example 8 Subcutaneous Xenograft Cancer Model

Human mammary xenograft, MaTu cells are maintained as adherent culturesin RPMI supplemented with 10% FBS. Ncr nude mice (8-12 weeks of age) areinoculated subcutaneously in the right flank with 5×10⁶ cells in 0.1 mLof 80% matrigel/20% HBSS. When tumors reach an average size of ˜180 mg(6 days), treatment is initiated. Antibodies are administered i.v. onceevery four days (Q4Dx3) at a dose of 10 mg/kg. Control mice are treatedwith PBS or an unconjugated monoclonal antibody. Daily examinations intothe health status of each animal are conducted. Each experimental groupconsists of 10 mice and the dosing volume was 0.1 mL/10 g body weight.The length and width of each tumor is measured by using an electroniccaliper 2-3 times per week and tumor weights (mg) are calculated basedon the formula of [length (mm)×width (mm)²]/2. All data, including dailyobservations, obtained throughout the course of the study aredocumented. Tumor growth inhibition (TGI) is calculated as 1−T/C×100,where T=final tumor weights from a treated group, and C=final tumorweights from the control group. The data demonstrates the therapeuticutility of antibodies for the treatment of tumors.

Other embodiments of the invention will be apparent to the skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

The invention claimed is:
 1. An antibody comprising a heavy chain and alight chain, each comprising one or more variable regions, wherein saidantibody is capable of binding an antigen or an epitope, and whereinsaid antibody comprises an amino acid linker which comprises one or moreprotease cleavage sites, wherein said protease cleavage sites areselected from the group consisting of SEQ ID NOS:26, 27, 28, 29, 30, 31,and 32, wherein the antibody comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:33-120.
 2. A kit comprising anantibody which comprises a heavy chain and a light chain, eachcomprising one or more variable regions, wherein the antibody is capableof binding an antigen or an epitope, and wherein the antibody comprisesan amino acid linker which comprises one or more protease cleavagesites, wherein the protease cleavage sites are selected from the groupconsisting of SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32, wherein theantibody comprises (a) a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein thelight chain comprises the amino acid sequence SEQ ID NO:81.
 3. A kitcomprising an antibody which comprises a heavy chain and a light chain,each comprising one or more variable regions, wherein the antibody iscapable of binding an antigen or an epitope, and wherein the antibodycomprises an amino acid linker which comprises one or more proteasecleavage sites, wherein the protease cleavage sites are selected fromthe group consisting of SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32,wherein the antibody comprises (a) a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein theheavy chain comprises the amino acid sequence SEQ ID NO:82.
 4. A kitcomprising an antibody which comprises a heavy chain and a light chain,each comprising one or more variable regions, wherein the antibody iscapable of binding an antigen or an epitope, and wherein the antibodycomprises an amino acid linker which comprises one or more proteasecleavage sites, wherein the protease cleavage sites are selected fromthe group consisting of SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32,wherein the antibody comprises (a) a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein thelight chain comprises the amino acid sequence SEQ ID NO:81 and the heavychain comprises the amino acid sequence SEQ ID NO:82.
 5. An antibodywhich comprises a heavy chain and a light chain, each comprising one ormore variable regions, wherein the antibody is capable of binding anantigen or an epitope, and wherein the antibody comprises an amino acidlinker which comprises one or more protease cleavage sites, wherein theprotease cleavage sites are selected from the group consisting of SEQ IDNOS:26, 27, 28, 29, 30, 31, and 32, wherein the antibody comprises (a) aheavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein thelight chain comprises the amino acid sequence SEQ ID NO:81.
 6. Anantibody which comprises a heavy chain and a light chain, eachcomprising one or more variable regions, wherein the antibody is capableof binding an antigen or an epitope, and wherein the antibody comprisesan amino acid linker which comprises one or more protease cleavagesites, wherein the protease cleavage sites are selected from the groupconsisting of SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32, wherein theantibody comprises (a) a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and, wherein theheavy chain comprises the amino acid sequence SEQ ID NO:82.
 7. Anantibody which comprises a heavy chain and a light chain, eachcomprising one or more variable regions, wherein the antibody is capableof binding an antigen or an epitope, and wherein the antibody comprisesan amino acid linker which comprises one or more protease cleavagesites, wherein the protease cleavage sites are selected from the groupconsisting of SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32, wherein theantibody comprises (a) a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH and (b) a light chainhaving the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and, wherein thelight chain comprises the amino acid sequence SEQ ID NO:81 and the heavychain comprises the amino acid sequence SEQ ID NO:82.
 8. The antibody ofclaim 5 which is conjugated to monomethylauristatin-E (MMAE).
 9. Theantibody of claim 6 which is conjugated to monomethylauristatin-E(MMAE).
 10. The antibody of claim 7 which is conjugated tomonomethylauristatin-E (MMAE).
 11. A pharmaceutical compositioncomprising the antibody of claim
 5. 12. A pharmaceutical compositioncomprising the antibody of claim
 6. 13. A pharmaceutical compositioncomprising the antibody of claim
 7. 14. The pharmaceutical compositionof claim 11, wherein the antibody is conjugated tomonomethylauristatin-E (MMAE).
 15. The pharmaceutical composition ofclaim 12, wherein the antibody is conjugated to monomethylauristatin-E(MMAE).
 16. The pharmaceutical composition of claim 13, wherein theantibody is conjugated to monomethylauristatin-E (MMAE).
 17. A kitcomprising: an antibody comprising one or more variable regions, whereineach of the one or more variable regions comprises a heavy chain and alight chain and is capable of binding an antigen or an epitope, andwherein the antibody comprises an amino acid linker which comprises oneor more protease cleavage sites; and solutions for suspending or fixingthe cells, detectable labels, solutions for rendering a polypeptidesusceptible to the binding of an antibody, solutions for lysing cells,and/or solutions for the purification of polypeptides wherein theantibody comprises a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a light chain havingthe structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein the lightchain comprises the amino acid sequence SEQ ID NO:81.
 18. A kitcomprising: an antibody comprising one or more variable regions, whereinsaid antibody binds one or more antigens or epitopes; and solutions forsuspending or fixing the cells, detectable labels, solutions forrendering a polypeptide susceptible to the binding of an antibody,solutions for lysing cells, and/or solutions for the purification ofpolypeptides wherein the antibody comprises a heavy chain having thestructure NH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a lightchain having the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and whereinthe heavy chain comprises the amino acid sequence SEQ ID NO:82.
 19. Thekit of claim 18, wherein the light chain comprises the amino acidsequence SEQ ID NO:81.
 20. An antibody comprising one or more variableregions, wherein said antibody binds one or more antigens or epitopes,wherein said variable region comprises a heavy chain having thestructure NH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a lightchain having the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and whereinsaid antibody comprises a variable region light chain comprising theamino acid sequence SEQ ID NO:81.
 21. An antibody comprising one or morevariable regions, wherein said antibody binds one or more antigens orepitopes, wherein said variable region comprises a heavy chain havingthe structure NH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a lightchain having the structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and whereinsaid antibody comprises a variable region heavy chain comprising theamino acid sequence SEQ ID NO:82.
 22. The antibody of claim 21, whereinsaid antibody comprises a variable region light chain comprising theamino acid sequence SEQ ID NO:81.
 23. The antibody of claim 20, which isconjugated to monomethylauristatin-E (MMAE).
 24. The antibody of claim21, which is conjugated to monomethylauristatin-E (MMAE).
 25. Theantibody of claim 22, which is conjugated to monomethylauristatin-E(MMAE).
 26. A pharmaceutical composition, comprising an antibody incombination with a pharmaceutically acceptable carrier, wherein theantibody comprises one or more variable regions, wherein said antibodybinds one or more antigens or epitopes, wherein said variable regioncomprises a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a light chain havingthe structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein saidantibody comprises a variable region light chain comprising the aminoacid sequence SEQ ID NO:81.
 27. A pharmaceutical composition, comprisingan antibody in combination with a pharmaceutically acceptable carrier,wherein the antibody comprises one or more variable regions, whereinsaid antibody binds one or more antigens or epitopes, wherein saidvariable region comprises a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a light chain havingthe structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH, and wherein saidantibody comprises a variable region heavy chain comprising the aminoacid sequence SEQ ID NO:82.
 28. The pharmaceutical composition of claim27, wherein the antibody comprises a variable region light chaincomprising the amino acid sequence SEQ ID NO:81.
 29. The pharmaceuticalcomposition of claim 26, wherein the antibody is conjugated tomonomethylauristatin-E (MMAE).
 30. The pharmaceutical composition ofclaim 27, wherein the antibody is conjugated to monomethylauristatin-E(MMAE).
 31. The pharmaceutical composition of claim 28, wherein theantibody is conjugated to monomethylauristatin-E (MMAE).
 32. Theantibody of claim 1, wherein said antibody is conjugated to atherapeutic or cytotoxic agent.
 33. The antibody of claim 32, whereinsaid antibody is conjugated to an agent selected from the groupconsisting of monomethylauristatin-E (MMAE), aplidin, azaribine,anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan,calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine,celebrex, chlorambucil, cisplatin, irinotecan (CPT-I1), SN-38,carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide,epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide,etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil,fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine,mechlorethamine, medroprogesterone acetate, megestrol acetate,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone,procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin,tamoxifen, taxanes, taxol, testosterone propionate, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade,vinblastine, vinorelbine, vincristine, ricin, abrin, ribomiclease,onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin, and functional analogs thereof.
 34. Apharmaceutical composition comprising a therapeutically effective amountof an antibody of claim 1 in combination with a pharmaceuticallyacceptable carrier.
 35. The pharmaceutical composition of claim 34further comprising one or more pharmaceutical agents.
 36. A kitcomprising an antibody of claim
 1. 37. The kit of claim 36, furthercomprising solutions for suspending or fixing the cells, detectablelabels, solutions for rendering a polypeptide susceptible to the bindingof an antibody, solutions for lysing cells, and/or solutions for thepurification of polypeptides.
 38. The kit of claim 37 wherein theantibody comprises a heavy chain having the structureNH₂-V_(H)1-linker-V_(H)2-C_(H)1-CH2-CH3-COOH; and a light chain havingthe structure NH₂-V_(L)1-linker-V_(L)2-CL-COOH.